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*Liver Diseases
The Journal of Immunology, 2006, 176: 6235-6244.
Copyright © 2006 by The American Association of Immunologists

Fas Ligand Is Responsible for CXCR3 Chemokine Induction in CD4+ T Cell-Dependent Liver Damage1

Michael W. Cruise*,{dagger}, John R. Lukens*,{dagger}, Aileen P. Nguyen*, Matthew G. Lassen*,{dagger}, Stephen N. Waggoner*,{dagger} and Young S. Hahn2,*,{dagger},{ddagger}

* Beirne Carter Center for Immunology Research, {dagger} Department of Microbiology, and {ddagger} Department of Pathology, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4+ T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core x TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4+ T cells into the liver of the immunized core+ TCR mice but not their core– TCR littermates. Strikingly, the inflammatory process in the liver of core+ TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-{gamma} production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3+ lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-{gamma} and cellular infiltration into the liver. These findings suggest that activated CD4+ T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Viral infection, including that of hepatitis C virus (HCV),3 is associated with development of chronic liver disease, which is characterized by massive T cell infiltration into the liver. In the liver of chronic HCV patients, the intrahepatic T cells have an activated Th1 phenotype and are effector cells (1). These liver-infiltrating T cells are responsible for damaging the liver and causing chronic liver disease rather than resolving HCV infection. The detailed molecular mechanism for T cell-mediated liver damage has yet to be identified; although the profound activated state of the liver infiltrate is involved in inducing Ag-independent killing of hepatocytes. Of particular interest is the correlation of these infiltrating T lymphocytes and the resulting cytokine/chemokine milieu within the liver. Apparently, chemokines secreted by the liver result in the recruitment of specific inflammatory cell subsets. Thus, studies aimed at identifying the mechanisms responsible for recruiting proinflammatory cells offer insight into the process of hepatic damage.

Besides the ability of the liver to process and detoxify the blood from toxins, the liver can trap and subsequently deactivate or delete activated T cells from the body (2, 3). The trapping of activated T cells induces the production of chemokines and adhesion molecules, resulting in the recruitment of inflammatory cells including macrophages, dendritic cells, neutrophils, NK, and activated T cells to the liver compartment. Once in the liver compartment, the activated immune cells are responsible for the destruction of liver parenchyma cells in bystander killing. Effector molecules, particularly Fas ligand (FasL), displayed on activated T cells are thought to play a crucial role in initiating hepatic damage. Indeed, the liver sinusoidal cells as well as hepatocytes constitutively express Fas and become sensitive to transmit Fas-mediated signaling upon encounter with FasL displayed on activated T cells.

It has been well documented that binding of FasL to the Fas receptor typically induces apoptosis (4). However, Fas engagement leads to enhanced proliferation of activated T cells, fibroblasts, and some tumor cells (5, 6, 7). Several studies have reported that FasL is involved in the induction of hepatic inflammation and the acceleration of liver regeneration observed after partial hepatectomy (8). In addition to this nonapoptotic effect of FasL, there is evidence for a novel role of FasL in inducing chemokine production. To this end, Fas signaling has been reported to play a role in the production of neutrophil attracting chemokines, KC and MIP-2 (9). Thus, the induction of chemokines by Fas signaling could provide a pathway to recruit immune cells to sites of inflammation and induce antipathogen responses.

Chemokines are small molecules responsible for the recruitment and localization of inflammatory cells to sites of tissue damage or infection. Each chemokine interacts with one or more receptors expressed on various cell types. Among the chemokine receptors, CXCR3, CCR5, CXCR4, and CCR3 have been demonstrated to play a role in inducing hepatic damage. CXCR3 is expressed on CD4+ T cells (activated, memory, Th1 type), CD8+ T cells (naive, activated, memory), NK, and B cells (10, 11) and binds to three different ligands: IFN-inducible protein 10 (IP-10)/CXCL10, monokine induced by IFN-{gamma} (Mig)/CXCL9, and IFN-inducible T cell {alpha} chemoattractant (ITAC)/CXCL11. These CXCR3 ligands are produced by several sources including monocytes, macrophages, endothelial cells, hepatocytes, and T cells in exposure to IFN-{gamma} (12, 13). CCR5 is expressed on Th1-type T cells, B cells, and binds to MIP-1{alpha}, MIP-1beta, and RANTES (14). The expression of CXCR4 is found on T cells, B cells, as well as monocytes/macrophages and binds the stromal cell-derived factor 1 (CXCL12) chemokine (15). In contrast to the chemokine receptors described above, CCR3 binds to eotaxin, RANTES, and MIP-1{alpha} and is expressed on cells involved in allergic reactions (i.e., Th2 lymphocytes, eosinophils, basophils, mast cells) (16). Thus, the distinct chemokine produced by various cell types plays a pivotal role in selective recruitment of specific cells to the site of inflammation.

Despite the fact that HCV replication occurs mainly in hepatocytes, immune cells have been reported to be susceptible for HCV infection (17, 18, 19). We have previously reported that the expression of HCV core protein in CD4+ T cells leads to altered phenotypes. Using the DO11.10 x HCV core double-transgenic mice, we demonstrated that the HCV core protein increases the proliferative potential of CD4 cells leading to activated-induced cell death after Ag stimulation. Additionally, HCV core increases FasL expression on these cells leading to the induction of hepatic inflammation and liver damage. In this study, we determined the mechanism(s) responsible for recruitment of liver lymphocytes and initiation of liver inflammation. To this end, we analyzed specific intrahepatic infiltrates and examined the kinetics of chemokine and chemokine receptors during acute hepatitis. This analysis revealed increased expression of CXCR3 and its chemokines. CXCR3-expressing cells were localized to areas of liver damage and the treatment of core+ TCR mice with anti-CXCR3-blocking Abs resulted in the reduction of hepatic damage. Importantly, blockade of Fas/FasL interaction revealed decreased cellular infiltrate and chemokine expression. This was confirmed by both in vivo and in vitro treatment with an agonist anti-Fas Ab that results in increased expression of CXCR3 ligands. Ultimately, this work demonstrates the role of Fas ligation in the induction of chemokine expression for the further recruitment of inflammatory cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Transgenic mice expressing full-length HCV core protein under the control of the CD2 promoter were bred with DO11.10 mice (The Jackson Laboratory). Progeny tail tissue samples were screened for the presence of HCV core DNA by PCR and confirmed by Western blot (20). The presence and absence of core expression in DO11.10 TCR mice are designated as core+ TCR and core– TCR, respectively. All mice were bred in a pathogen-free facility at the University of Virginia (UVA) and routinely tested for mouse hepatitis virus, Helicobacter, and other pathogens. Mice were treated as previously described (21) with 25 nM of the OVAII peptide (ISQAVHAAHAEINEAGR, OVA323–339) or irrelevant I-Ad-restricted peptide (ASFEAQGALANIAVDKA, I{alpha}52) (UVA Biomolecular Research Facility) screened for endotoxin, dissolved in DPBS, and injected in a volume of 250 µl. All mice were handled according to protocols approved by the UVA Institutional Animal Care and Use Committee.

Abs and flow cytometry

Monoclonal anti-mouse CD3{epsilon}, CD4, CD8, CD11c, CD44, DX5, F4/80, CD95L, Fc{gamma}R III/II (Fc block) and functional grade anti-FasL, anti-Fas, and appropriate isotype Abs were purchased from eBioscience. Anti-mouse CXCR4, CCR5, and CCR3 were purchased from BD Biosciences and CXCR3 from Zymed Laboratories. KJ1-26, streptavidin-FITC and PE, and appropriate isotype control Abs were purchased from Caltag Laboratories. Additionally, biotin and PE donkey F(ab')2 anti-rabbit, anti-rat, and anti-goat secondary Abs were purchased from Jackson ImmunoResearch Laboratories. Purified anti-mouse Mig/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 were purchased from R&D Systems and anti-mouse CD3 was obtained from Santa Cruz Biotechnology.

Preparation of splenocytes and liver cells

Splenocytes were prepared by mechanical disruption and isolation over a Ficoll gradient. Nonparenchyma liver cells were isolated as previously described (21). Briefly, the liver sections were finely minced and placed in a digestion mix at 37°C for 20 min. Hepatocytes and debris were removed after collagenase digestion by centrifugation at 30 x g for 3 min at 4°C. The supernatant was removed, cells were collected, and resuspended in 22% Nycodenz (Sigma-Aldrich). Then Iscoves’s was underlaid with the cell/Nycodenz mixture and separated at 1500 x g for 20 min at 4°C. The interface of the gradients were collected and viable cells were counted using trypan blue. Cells were resuspended and blocked with Fc block followed by incubation with isotype controls or primary Abs. Detection of chemokine receptors was performed using purified primary Abs followed by incubation with biotin-labeled secondary and streptavidin-PE. Data from flow cytometry experiments were analyzed with FlowJo software (Tree Star). The total number of positive cells was calculated as the percentage of gated cells multiplied by the total number of viable cells.

Administration of anti-FasL and anti-CXCR3 Ab

Core+ TCR mice were treated with anti-FasL Ab by injecting 100 µg of anti-FasL (MFL3) Ab or isotype control at the time of OVAII stimulation. In separate experiments, mice were treated with anti-CXCR3 Ab. The polyclonal anti-CXCR3 was raised in rabbit against the NH2 portion (NH2-PYDYGENESDFSDSPP-COOH) of the CXCR3 as previously reported (22). The Ab was produced by Biosource International and was desalted. Then anti-CXCR3 or prebleed was i.p. injected into core+ TCR mice at the time of OVAII stimulation.

Histological evaluation and immunohistochemistry (IHC)

Tissue samples were fixed in 10% buffered formalin solution for 24 h and then embedded in paraffin. Liver damage was evaluated by conventional H&E staining, TUNEL assay, and Ki67 staining; all were performed as previously reported (21). The detection of Mig/CXCL9, IP-10/CXCL10, ITAC/CXCL11, CD3, B220, macrophage (F4/80), and CXCR3 were performed in formalin-fixed paraffin-embedded (FFPE) tissue using methods previously reported (21). Ag retrieval for Mig/CXCL9, IP-10/CXCL10, ITAC/CXCL11, and CXCR3 was performed by microwave irradiation in citrate buffer (pH 6), however, Ag retrieval for B220 and CD3 staining was performed in EDTA buffer (pH 8), while Ag retrieval for F4/80 staining was performed with a proteinase K digest at room temperature for 30 min.

The specificity of Mig/CXCL9 and IP-10/CXCL10 Abs was tested by incubating the primary Ab with recombinant mouse Mig/CXCL9 or IP-10/CXCL10 (PeproTech). The rMig/CXCL9 was able to block Mig/CXCL9 staining but not IP-10 staining, while rIP-10 was able to block IP-10/CXCL10 staining but was unable to block Mig/CXCL9 staining. Quantitation of chemokine staining was performed with ImageJ software. Briefly, five random x40 images from the same tissue section were acquired and the percent of total area staining positive was calculated. A minimum of five animals per group was included in the analysis.

RNase protection assay (RPA)

Livers and spleen sections were collected, then RNA extracted with TRIzol (Invitrogen Life Technologies) and stored at –80°C. RPA was performed per manufacturer’s instructions (BD Biosciences). Briefly, a custom probe set containing available cDNA of interest was synthesized in the presence of [32P]UTP yielding labeled RNA probes. The resulting product was treated with DNase to remove residual cDNA. The RNA and probes were precipitated with ammonium acetate and ethanol, then 10 µg of RNA was hybridized overnight at 56°C with 3.95 x 105 cpm/µl probe. Hybridized samples were treated with RNase followed by deactivation of RNase with proteinase K. The resulting protected probes were precipitated as above and the samples resolved on an acrylamide gel and analyzed by autoradiography as well as phosphoimaging (performed with Packard Cyclone Storage Phosphoimager). Samples were normalized to L32 and GAPDH.

In vivo and in vitro ligation of the Fas Receptor

Core– TCR mice were i.v. injected with 2 µg of anti-Fas (Jo2) Ab and tissue samples were collected at various time points. Mouse macrophage (RAW267.4) and hepatocyte lines (Hepa 1–6) were obtained from American Type Culture Collection (ATCC). For chemokine determination, macrophages and hepatocytes were seeded at 3 x 105 cells/ml and 2 x 105 cells/ml, respectively, in a 24-well plate and then allowed to adhere overnight. The medium were replaced and cells were treated with 10 ng/ml LPS (Sigma-Aldrich) or 100 IU of IFN-{gamma} (R&D Systems) and 1 µg/ml anti-Fas (Jo2) or an isotype control Ab. Cells were treated for various times and the supernatant was collected for chemokine determination.

IP-10/CXCL10 ELISA

ELISA specific to murine IP-10 was purchased from R&D Systems. It was used per the manufacturer’s instructions for the determination of the chemokines in tissue culture supernatants.

Statistical analysis

A paired Student’s t test or ANOVA was used to evaluate the significance of the differences. Statistical analysis was performed with SPSS version 11.5, with a value of p < 0.05 regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Liver-infiltrating CD4+ T cells are responsible for recruitment of innate immune cells to the liver

In this study, we use the HCV core x DO11.10 double-transgenic mice in which we can study the effects of HCV core on the function of T lymphocytes under Ag-specific stimulation. Using this model, we have previously demonstrated that DO11.10 transgenic CD4+ T cells are specifically recruited to the liver but not to other organs following OVAII peptide injection (21). In addition, increased expression of FasL on CD4+ T cells was associated with liver inflammation and hepatic damage. To determine an underlying mechanism for the role of FasL-expressing CD4+ T cells in liver inflammation, we first analyzed the infiltration of specific cell types into the liver following CD4+ T cell activation. To this end, we administered 25 nM OVAII peptide into both core+ TCR transgenic and core– TCR littermate mice and examined the accumulation of inflammatory cells such as T cells (DX5–CD3+), NK (DX5+CD3–), NKT (DX5+CD3+), Kupffer cells (F4/80+), and dendritic cells (CD11c+ high) in the liver by flow cytometry.

As shown in Fig. 1, massive lymphocytic infiltration was observed in core+ TCR mice following antigenic stimulation of transgenic CD4+ T cells. Increased infiltration of CD3+ T cells was found in the liver after OVAII injection (Fig. 1A). In contrast, no change in liver-infiltrating lymphocytes was detectable in core+ TCR mice after irrelevant peptide injection nor did lymphocytes from the spleen of these mice express increased activation markers (data not shown). Importantly, increases in liver-infiltrating CD3+ T cells were accompanied by increased numbers of NK (Fig. 1B) and NKT cells (Fig. 1C). Furthermore, there was an influx of APCs with increased numbers of dendritic (Fig. 1D) and Kupffer cells (Fig. 1E). Immunohistochemical analysis revealed that dendritic cells were primarily localized around portal regions with decreased numbers of dendritic cells in closer proximity to the central vein (data not shown). In contrast, F4/80+ cells, primarily Kupffer cells, reside between the portal and central vein and are increased at sites of inflammation in close contact with the infiltrating lymphocytes (data not shown). Taken together, these data suggest that liver-infiltrating CD4+ T cells are responsible for induction of liver inflammation and recruitment of innate immune cells. The recruitment of the CD4 lymphocytes into the liver resembles the recruitment of CD8 cells both in the OT-1 system and the adoptive transfer system (21, 23).


Figure 1
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  		FIGURE 1. Increased numbers of innate immune cells accompanied by liver-infiltrating T cells. Core+ TCR and littermate mice were injected i.v. with OVAII peptide. At various time points after OVA II injection, liver-infiltrating cells were collected, enumerated, and their subtypes of viable cells were determined by flow cytometry. Total cell numbers where calculated per animal with the means and SEM for T cells (A), NK cells (B), NKT cells (C), dendritic (CD11c+) cells (D), and Kupffer (F4–80+) cells (E) are shown for both core+ TCR and core– TCR littermate mice. Each group contains a minimum of five mice per time point (*, p < 0.05; **, p < 0.001).

 
Liver-infiltrating lymphocytes express CXCR3

To determine whether distinct subsets of peripheral immune cells are selectively recruited to the liver, we first examined the expression of chemokine receptors in liver-infiltrating lymphocytes. Flow cytometry analysis on viable cells reveals that the proportion of cells positive for CXCR3 was increased in the intrahepatic infiltrates (Fig. 2A). There was minimal alteration in the number of cells expressing CCR5, CXCR4, or CCR3 as compared with CXCR3-positive lymphocytes.


Figure 2
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  		FIGURE 2. CXCR3+ lymphocytes are predominantly recruited to the liver. A, Liver lymphocytes were analyzed by flow cytometry for expression of CXCR3, CCR5, CCR3, and CXCR4. The total number of viable liver lymphocytes expressing different chemokine receptors was determined per mouse. The expression of CXCR3 in CD3+ T cells, NK, and NKT cell populations shows a different kinetic of infiltration (B). Although CCR5 (C), CCR3 (D), and CXCR4 (E) are expressed by intrahepatic cells, their populations represent a reduced proportion of the cellular infiltrate. Each group contains five mice per time point and the mean values and the SEM are displayed in the graphs.

 
We further analyzed the expression of specific chemokine receptors on distinct lymphocyte subsets (i.e., CD3, NK, NKT cells) by double staining for isolated intrahepatic lymphocytes with Abs for CD3, DX5, as well as chemokine receptors. Cells were gated on T (CD3+DX5–), NK (CD3–DX5+), and NKT (CD3+DX5+) cell populations. Interestingly, various CXCR3+ lymphocyte subsets migrated into the liver with different kinetics (Fig. 2B). Specifically, a maximal increase in CXCR3+ NK and NKT cells were detectable on days 1 and 2 while the number of CXCR3+CD3+ cells reached maximal levels at days 3 and 5 after CD4+ TCR activation. Although there were slight increases in the number of CCR5+ and CCR3+ cells on days 1 and 2, CXCR4+ cells reached their maximal numbers around day 3 after OVAII injection. Flow cytometry analysis also demonstrates a significantly higher proportion of CXCR3+ cells in the liver than the spleen (data not shown). Both the increased cell number and high percentage of CXCR3+ cells in the liver suggest that CXCR3+ lymphocytes are recruited to the liver through specific mechanisms.

Blockade of CXCR3+ cell migration prevents intrahepatic infiltration and hepatic damage

Although a dramatic increase in the number of CXCR3+ lymphocytes was detected in the liver by flow cytometry, it is not known whether they are localized to the site of hepatic damage. Thus, we sought to determine the localization of the infiltrating cells within the liver and whether they localized to areas of hepatic damage. CXCR3 was detected in FFPE tissue with specific staining of CXCR3 on the cell surface of liver-infiltrating mononuclear cells (Fig. 3A). Staining for CXCR3 in splenic sections revealed that CXCR3+ cells were primarily restricted to the T cell zones and were in greatest number at day 1 with decreasing expression at later time points (Fig. 3C). IHC labeling for CXCR3 within the liver confirmed the increased infiltration of CXCR3+ cells into the liver of core+ TCR mice (Fig. 3D). The CXCR3+ cells primarily localized to the periportal area early after antigenic stimulation then migrate to the lobular area later during the inflammatory process. Importantly, these CXCR3-expressing cells demonstrated organization into lymphoid aggregates (Fig. 3, A and D) correlating with hepatic damage and hepatocellular death.


Figure 3
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  		FIGURE 3. CXCR3+ cells are localized in areas of liver inflammation. Liver and spleen tissue sections were harvested and stained with anti-CXCR3 Ab. Cells showing the red color, indicated by the arrow, represent a positive stain for CXCR3 localized to the cell surface of infiltrating lymphocytes in the liver (A), while detection Ab alone demonstrated no staining (B). (Original magnification, x200). Spleen sections were stained at various times after Ag stimulation, demonstrating decreasing staining of CXCR3 (C). The majority of liver-infiltrating lymphocytes are CXCR3+ (D). Additionally, the CXCR3+ cells are localized into lymphoid aggregates, indicated by the arrows. The displayed images are representative of a minimum of five mice per group per time point.

 
To verify the role of these CXCR3+ cells in the induction of hepatic damage, we attempted to block the CXCR3 engagements with its ligand. Because CXCR3 can bind and be activated by Mig, IP-10, and ITAC, we chose to treat our mice with a blocking Ab against CXCR3 instead of Abs against all three chemokines. The core+ TCR mice were treated with polyclonal or prebleed IgG at the time of Ag stimulation followed by histological studies performed 3 days later. Treatment of the core+ TCR mice with blocking Abs to CXCR3 reduced hepatic damage and inflammation (Fig. 4). As evident by H&E staining, there was less lymphocyte infiltration into the liver of core+ TCR mice treated with anti-CXCR3 Ab and no formation of lymphoid aggregates around the portal triads or in the lobular regions (Fig. 4A). This observation is confirmed by CD3 staining in the liver (Fig. 4B).


Figure 4
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  		FIGURE 4. Treatment with anti-CXCR3 Ab reduces hepatic damage. Mice were treated with anti-CXCR3 Ab at the time of OVAII peptide injection. Liver and spleen tissues were harvested on day 3 after anti-CXCR3 Ab treatment. Treatment of animals with anti-CXCR3 Ab reduces the cell infiltration as demonstrated in H & E staining (A) and CD3 immunostaining (B). Additionally, Ki67 immunostaining of hepatocytes (C) and decreased ALT levels (D) revealed reduced hepatic damage. Sections are representative of five animals per group. The results of this experiment were reproducible in two other independent experiments.

 
Hepatocytes are mostly quiescent but proliferate in the presence of hepatic damage invoking the regeneration response. Thus, an increase in Ki67+ staining of hepatocytes has been reported to indicate an increase in hepatocellular death, whereby the surrounding hepatocytes regenerate to replace the dead or dying cells. Indeed, treatment with anti-CXCR3 Ab abrogates hepatic damage as evident with significantly lower numbers of Ki67+ hepatocytes (Fig. 4C). The reduced hepatic damage was also confirmed by TUNEL assay (data not shown). Additionally, treatment of the mice with the anti-CXCR3 Ab reduced the mean serum alanine aminotransferase (ALT) levels. The mean ALT level in core– TCR mice was 27 U/L while the core+ TCR and the core+ TCR treated with prebleed serum were 140 and 81 U/L, respectively. The treatment with anti-CXCR3 Ab reduced the mean serum ALT in core+ TCR to 27 U/L. Importantly, Ki67 staining and flow cytometry staining for activation markers were not altered in the spleen, indicating that anti-CXCR3 Ab treatment did not interfere with Ag stimulation of T lymphocytes (data not shown). Taken together, the blockade of CXCR3 infiltration by anti-CXCR3 Ab treatment prevented hepatic inflammation and resulting hepatic damage.

Increased expression of CXCR3 ligands, IP-10, and Mig in the liver

Although infiltration of CD3+ and CD4+ T cells into the liver correlates with recruitment of innate immune cells including NK and NKT cells, it is not clear whether these cells migrate from other peripheral lymphoid sites or undergo in situ proliferation in the liver. Given, the increased migration of CXCR3+ lymphocytes to the liver of core+ TCR mice following CD4+ activation, it is likely that the hepatic environment of core+ TCR mice generates enhanced production of CXCR3 ligands to recruit inflammatory cells.

To test this possibility, we examined the production of chemokines in core+ TCR mice following OVAII injection. First, we analyzed the induction of chemokine-specific mRNA in the liver by RPA (Fig. 5A). After Ag stimulation, core+ TCR mice demonstrated dramatically increased intrahepatic mRNA for IP-10. Additionally, there was increased mRNA expression of KC and MCP-1, both of which were previously reported to be elevated during Fas-mediated inflammation (9, 24). Consistent with prior observations (Fig. 2), there was a detectable increase of CXCR3 mRNA in the hepatic compartment. Importantly, there was no increase in mRNA expression of other chemokines except minor increase in MIP-3{alpha} and RANTES mRNA level. In contrast to the significantly elevated mRNA levels of IP-10, CXCR3, KC, and MCP-1 in core+ TCR mice, core– TCR mice demonstrated increased levels of RANTES and MIP-3{alpha} (data not shown). The KC chemokine is thought to be the mouse homolog of IL-8 and recruits neutrophils, while MCP-1 primarily recruits macrophages but can also recruit and activate stellate cells (25). Of note, IP-10 is a ligand for CXCR3 and the cells infiltrating the liver are primarily CXCR3 positive, suggesting that the increased IP-10 mRNA might be responsible for the recruitment of CXCR3+ lymphocytes to the liver of core+ TCR mice.


Figure 5
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  		FIGURE 5. Increased expression of CXCR3-specific chemokines in the liver of core+ TCR mice. A, mRNA levels of chemokines were determined in whole liver lysates from core+ TCR mice by RPA and normalized to day 0. The increased expression of KC, MCP-1, and IP-10 were detectable in these mice after Ag stimulation. The expression of the chemokines was analyzed by IHC (B and C) and quantified using ImageJ software (D and E). The specificity of anti-chemokine Abs was tested by competing the Ab with recombinant protein IP-10 or Mig (B). Liver sections were stained for Mig (C) and red precipitate indicates a positive stain (original magnification, x40). Using the ImageJ software, the staining patterns were analyzed by quantifying the percentage of area positive for chemokine staining in the sections. Core+ TCR mice demonstrate a significant increase (***, p < 0.0001; *, p < 0.01) in both Mig and IP-10 expression. High magnification (original magnification, x200) demonstrates that as time progresses after Ag stimulation, the detection of Mig cytoplasmic staining (F) changes from primarily endothelial cells on day 1 to mononuclear cells and hepatocytes on days 2 and 3. The mean values and the SEM represent a minimum of five mice per group, per time point, and are displayed in the graphs.

 
To confirm whether increased mRNA expression of CXCR3 ligands correlates to the production of functional CXCR3 chemokines, we examined the protein expression of CXCR3 ligands by immunohistochemistry. Because CXCR3 binds three chemokines (IP-10, Mig, and ITAC), we attempted to assess the production of all three chemokines. FFPE tissues from core+ TCR and core– TCR mice were stained with Abs specific for Mig or IP-10 or ITAC. To verify the specificity of the Abs, the primary anti-Mig Ab was incubated with rMig protein which was able to abrogate staining while incubation with rIP-10 was unable to inhibit staining (Fig. 5B). The anti-IP-10 Ab demonstrated reciprocal specificity (data not shown). Staining core+ TCR and core– TCR mice for IP-10 and Mig revealed significantly increased expression of these chemokines in the core+ TCR mice as compared with core– TCR littermates (Fig. 5C). Quantitative analysis for IHC staining with ImageJ software confirmed that mice expressing the HCV core protein demonstrate increased protein expression of Mig (Fig. 5D) and IP-10 (Fig. 5E) in the liver as compared with their core– TCR littermates.

In contrast to the staining of Mig and IP-10, which demonstrate chemokine expression by multiple cell types, ITAC expression was more restricted. Immunostaining of ITAC demonstrated expression limited to cells that appear morphologically to be dendritic cells and macrophages (data not shown). However, only a very few ITAC-positive cells could be detected in the liver and only early, day 1, after antigenic stimulation. Additionally, microscopic analysis revealed intriguing expression kinetics of CXCR3 ligands in which the cellular source of chemokine expression changed over time (Fig. 5F). Although initial expression of the chemokines was primarily restricted to liver sinusoidal endothelial cells, the expression expanded to liver mononuclear cells and hepatocytes, which become the major source of chemokine expression later in the inflammatory process. The change in the cellular source of chemokine protein expression may explain the kinetic discrepancy between the protein and mRNA expression. The delay in protein expression (detection) may also be due to posttranslational modifications.

FasL expression by CD4+ T cells induces the expression of CXCR3 ligands by liver parenchyma cells

Recently, a novel function for FasL has been reported in the induction of chemokine expression and acceleration of the inflammatory response (26). Therefore, the enhanced expression of FasL on HCV core-expressing CD4+ T cells (21) may implicate the role of Fas/FasL interaction in inducing chemokine expression. To determine the role of Fas ligation on chemokine production, core+ TCR mice were treated with blocking Abs to FasL to prevent Fas/FasL engagement. As shown in Fig. 6A, there was a significant decrease in the number of lymphocytes infiltrating the liver after treatment with anti-FasL. This decrease in infiltration is accompanied by a decrease in liver inflammation and hepatocellular death. Additionally, the inhibition of Fas ligation reduced the level of CXCR3 ligand production. RPA analysis of whole liver lysates reveals that blocking Fas ligation significantly reduces the amount of IP-10 mRNA detected (Fig. 6, B and C), p < 0.02. The results of the RPA were normalized to the housekeeping gene L32 and the mean was displayed in Fig. 6B. In addition, IHC revealed reduced Mig staining in mice receiving anti-FasL treatment, which was confirmed by quantitative ImageJ analysis for Mig (Fig. 6, D and E) and IP-10 (data not shown). As a complementary approach, liver-infiltrating lymphocytes for core+ TCR mice were adoptively transferred into Fas-deficient lpr mice backcrossed into the BALB/c genetic background. The results of these experiments reveal that lpr mice receiving core+ TCR liver lymphocytes did not induce Mig production to the same level as the age-matched BALB/c mice receiving cells from the same source (data not shown). Taken together, these results confirm that the increased expression of FasL by HCV core-expressing CD4+ T cells and Fas ligation plays a role in the induction of CXCR3.


Figure 6
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  		FIGURE 6. Blockade of Fas/FasL interaction reduces chemokine expression and liver inflammation. Core+ TCR and core– TCR littermate mice were treated with 100 µg of anti-FasL Ab at the same time as OVAII injection. The influx of lymphocytes into the liver was significantly reduced in the core+ TCR mice treated with anti-FasL (A). Additionally, this reduced cellular infiltrate corresponded to decreased mRNA expression of IP-10 (B and C) as measured by RPA. The cells were isolated from the liver and the IP-10 RPA results were normalized to housekeeping gene (L32) expression (B). The protein expression of Mig (D and E) as measured by IHC was also reduced. Samples were isolated 3 days after last stimulation. The mean values and the SEM are displayed in the graphs. For A, D, and E, the number of mice per treatment was 4 while the number of mice per treatment for B and C is 3.

 
Fas ligation is directly responsible for enhancing the expression of CXCR3 ligand in IFN-{gamma}-stimulated hepatocytes and macrophages

To further evaluate a direct role of Fas signaling in the induction of CXCR3 ligands as well as specific cell types responsible for chemokine production upon Fas ligation, we established experiments to trigger the Fas-signaling pathway by exploiting a Fas cross-linking Ab. In this experimental setting, Fas cross-linking Ab mimics the engagement of Fas by FasL expressed on activated T cells. Using anti-Fas Ab treatment, we analyzed IP-10 and Mig levels by the direct ligation of the Fas receptor in vivo in an HCV core-independent system. Additionally, we further determined specific cell types responsible for the production of CXCR3 chemokines in vitro following Fas ligation.

We first examined the production of Mig and IP-10 in vivo by administering Fas cross-linking Ab (clone Jo2) into DO11.10 mice that neither express the HCV core protein nor have been activated by OVA antigenic stimulation. A titration study of anti-Fas Ab determined that an i.v. dose of 2 µg did not induce massive hepatotoxicity or apoptosis of hepatocytes (data not shown). Immunohistochemical analysis revealed significant increases in the expression of Mig (Fig. 7A) and IP-10 (data not shown). The production of Mig was initially detectable at 12 h after anti-Fas Ab treatment and continuously secreted at 24 and 48 h but slowly disappears 72 h after anti-Fas Ab treatment. However, PBS-injected mice did not demonstrate Mig expression after 12 h. These results confirm a direct role of Fas signaling in the induction of CXCR3.


Figure 7
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  		FIGURE 7. Fas engagement increases IP-10 and Mig production in vivo and in vitro. Administration of the agonist anti-Fas (Jo2) Ab to mice results in increased Mig expression in vivo (A). Mice were injected i.v. with PBS or 2 µg of anti-Fas (Jo2) Ab and liver tissue harvested from injected mice and immunostained for Mig. Elevated IP-10 production was demonstrated in vitro when (B) RAW264.7 (mouse macrophage) and (C) Hepa1–6 (mouse hepatocyte) were stimulated with 100 U/ml murine recombinant IFN-{gamma} or 10 ng/ml LPS in the presence of 1 µg/ml anti-Fas (Jo2) or an isotype control Ab. After treatment for 12 h (RAW264.7) or 24 h (Hepa 1–6), tissue culture supernatants were tested for IP-10 production. The mean values and the SEM are displayed in the graphs. The Student t test demonstrates statistical significance of p < 0.01.

 
We next examined specific cell types responsible for the production of IP-10. For this analysis, we chose hepatocytes and macrophages to assess the IP-10 production in vitro upon Fas ligation because these cells are shown to secrete CXCR3 ligands by in vivo IHC analysis (Fig. 5E). The resident and the infiltrating CD4 and NK lymphocytes secrete IFN-{gamma} (data not shown) and are suspected to be responsible for the induction of IFN genes such as CXCR3. Therefore, we treated the macrophage and hepatocyte cell lines with IFN-{gamma}. The treatment with anti-Fas Ab results in a 4-fold increase for IP-10 production by IFN-{gamma}-stimulated macrophages as compared with that in control Ab-treated or LPS-stimulated macrophages (Fig. 7B). The above trend was also observed in thioglycolate-elicited peritoneal macrophages. The increase in IP-10 production was ~150-fold greater than the baseline measurement. Similar results were obtained with the Hepa 1–6 mouse hepatocyte cell line (Fig. 7C). After 24 h of anti-Fas Ab treatment, there was a significant increase in the expression of IP-10 in IFN-{gamma}-stimulated hepatocytes as compared with the increase observed following treatment with only Jo2 or IFN-{gamma}. There was a 1.5-fold increase in IP-10 following dual treatment with IFN-{gamma} and Fas ligation as compared with IFN-{gamma} and isotype Ab, and an 8-fold increase in IP-10 expression as compared with baseline expression. Interestingly, the treatment of the macrophage and hepatocyte lines with IFN-{gamma} increases the surface expression of the Fas receptor (data not shown). The expression of chemokines requires activation of IFN-{gamma} pathways. These results further demonstrate that Fas ligation can result in increased expression of proinflammatory chemokines, specifically, CXCR3 ligands, in both macrophages and hepatocytes.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Typically Fas engagement on hepatocytes can lead to the induction of hepatocyte death through the association of Fas-associated death domain with caspase 8, leading to activation of downstream apoptotic pathways (4). However, recent evidence has reported that engagement of Fas plays other roles in the induction of the inflammatory response. Fas/FasL engagement of T lymphocytes can provide costimulatory signals (27) and the engagement on Fas-expressing cells induces the production of proinflammatory molecules (28, 29). FasL has also been demonstrated to induce chemokine production by various cell types, including PANC-1 cells in which Fas ligation induces MCP-1 and IL-8 (30). Indeed, the interaction of Fas/FasL has also been shown to play a role in the induction of lung damage and fibrosis (31) along with the induced production of KC and MIP-2 (9). More recently engagement of Fas on dendritic cells has been demonstrated to alter chemokine production and increase the production of MCP-1, RANTES, MIP-2, MIP-1{alpha}, and MIP-1beta chemokines (26). This would provide a mechanism for the recruitment of lymphocytes to sites of inflammation and the promotion of APC-T cell interactions.

In this report, we demonstrated the role of FasL in the induction of proinflammatory chemokine production and hepatic damage. Using double-transgenic mice that express HCV core in T lymphocytes of DO11.10 TCR mice, we observed increased infiltration of lymphocytes and innate immune cells (i.e., macrophage and dendritic cells) in the liver of core+ TCR mice following OVAII injection. The subsequent analysis of chemokine expression revealed the increased production of CXCR3-specific chemokines, IP-10 and Mig in core+ TCR mice. The increase in hepatic expression is correlated with an increase in CXCR3-expressing T cells, NK, and NKT cells found in the liver. Importantly, treatment with an anti-CXCR3 Ab resulted in decreased lymphocyte infiltration and hepatic damage. Blockade of Fas/FasL interaction with a neutralizing anti-FasL Ab demonstrated that HCV core-induced hepatic damage is mediated by bystander killing of hepatocytes through Fas/FasL interactions. The interaction of FasL-expressing lymphocytes could induce the production of chemokines by Fas-expressing cells. Indeed, these observations were confirmed by direct ligation of the Fas receptor. Taken together, these data suggest that Fas/FasL interactions provide a costimulatory signal that can act synergistically with IFN-{gamma} to increase production of CXCR3 ligands. The mechanism behind this synergistic effect has yet to be determined. We speculate that this effect is through the STAT system. Initial results suggest that the synergistic effect of Fas ligation and IFN-{gamma} might be independent of the alteration of STAT1 (pY701). However, STAT1 can also be phosphorylated at serine 727 and it is possible that the demonstrated synergistic effect of IFN-{gamma} and Fas uses the p38 pathway of STAT1 activation. Further work in this area will help us elucidate the point at which the IFN-{gamma} and Fas pathways interact.

The expression of CXCR3 has been reported to play a crucial role in development of several immunological disease states (e.g., systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, diabetes) (32, 33). In addition, CXCR3 plays a role in the progression of liver disease in chronic HCV patients. The mRNA levels of IP-10, Mig, and ITAC are increased in liver tissues derived from chronic HCV patients and the levels of enhanced expression of these chemokines correlated with severity of liver disease (34, 35, 36). The serum levels of IP-10 can also serve as a marker for the effectiveness of IFN-{alpha} and ribavirin treatment as the levels of IP-10 dramatically decrease in chronic HCV patients who respond to therapy (37). Additionally, the pretreatment levels of the CXCR3 ligands may have an effect on the outcome of HCV treatment, where increased levels of these chemokines before treatment corresponds with treatment failure (38). Taken together, the CXCR3 system may be an important target in HCV therapy to prevent hepatic damage and improve the effectiveness of antiviral treatments.

The CXCR3 binds three chemokines: ITAC, IP-10, and Mig. These ligands exhibit different binding affinity for the receptor with an order of ITAC > IP-10 > Mig (39). Interestingly, the affinity of these chemokines for CXCR3 exhibits an inverse relationship to the amount of chemokine production such that Mig with lowest affinity to CXCR3 shows the highest chemokine production (40). Our studies in this report demonstrate a similar pattern of protein expression of the CXCR3 ligands with the expression order of Mig > IP-10 >> ITAC. There are several cell types capable of producing CXCR3. In general, their expression is constitutively low, however, their activation can lead to widespread expression by multiple organs and cell types including endothelial cells, hepatocytes, and stellate (12, 13, 41). It is worthwhile to point out that the biological function of CXCR3 engagement varies depending on specific cell types. For examples, the engagement of CXCR3 on endothelial cells inhibits their proliferation, while the same engagement on CXCR3 activates stellate cells (42, 43). The activation of stellate cells within the hepatic compartment is of particular interest as they are the liver fibroblast cells. The constant high levels of CXCR3 in the presence of hepatic damage could therefore predispose the liver to the formation of liver fibrosis and liver dysfunction/cirrhosis.

In addition to the induction of chemokines by cytokines and infiltrating lymphocytes, the expression of HCV viral proteins by hepatocytes acts synergistically to induce CXCR3 (44). At first this appears paradoxical because CXCR3 would recruit cytolytic lymphocytes to the liver where the lymphocytes could destroy infected hepatocytes. However, HCV-infected hepatocytes are more resistant to killing by infiltrating lymphocytes (45, 46). As a result, the infiltrating lymphocytes destroy noninfected hepatocytes. The elimination of uninfected cells by the infiltrating cells thus allows a selective advantage for HCV-infected hepatocytes. In addition, the recruitment of activated cells to the liver leads to the bystander deletion of HCV-specific lymphocytes (47), which creates an environment tolerogenic to HCV Ags/infected cells.

The role of CXCR3 ligands during the inflammatory response extends beyond chemotaxis and the localization of cells to inflammatory sites. First, IP-10 and Mig exert direct antiviral activities (48) and the accumulation of IP-10 is also linked to an increase in IFN-{gamma} (49) and the activation of NK cells (50). Second, the production of CXCR3 ligands enhances the proliferation and effector function of lymphocytes in a IL-2-independent fashion (51), which further augments the cytotoxic potential of the infiltrating lymphocytes. Third, the production of chemokines and the ligation of CXCR3 are necessary for the production of long-term memory lymphocytes (52). In the case of chronic inflammatory conditions, the elevated production of chemokines would play a role in promoting and sustaining inflammation. The high levels of the chemokines expressed in the inflamed liver would induce the recruitment of lymphocytes where they can induce hepatic damage. The CXCR3 ligands would increase the activation status and effector functions of lymphocytes thereby amplifying the inflammatory responses and creating a proinflammation, pro-Th1 loop of infiltration and hepatic damage.

Our work in this report provides compelling evidence for the critical role of Fas/FasL in the induction of CXCR3 chemokines in hepatic damage. As such, inhibition of Fas/FasL interactions or Fas signaling might prove to be an important therapeutic prevention for hepatic inflammation and damage. One approach would be to block Fas signaling with a drug like suramin (Food and Drug Administration approved), which has proven useful in the inhibition of Fas-induced acute hepatitis (53, 54). We are currently investigating whether suramin is able to inhibit the production of CXCR3 by abrogating Fas signaling and whether this drug would be useful in the treatment of acute hepatitis as well as chronic liver inflammatory diseases. Another approach to reduce hepatic damage is to block the action of CXCR3. Several groups have developed small nonpeptide inhibitors that can bind to and inhibit the function of chemokine receptors in vitro and in vivo (40, 55). In this case, NBI-74330 (40) demonstrates the ability to inhibit receptor activation by all three CXCR3 ligands and this series (T487) of CXCR3 antagonists are currently in phase II clinical trials. In addition to manufactured inhibitors, four other natural CXCR3 antagonists have been reported to be effective in the blockade of CXCR3 action (56). Given the role that CXCR3 ligands play in induction of hepatic damage, prevention of CXCR3 engagement may be beneficial not only for the treatment of the acute/early infection period but also for treatment during the early phases of fibrosis.

In summary, we report here that Fas ligand induces CXCR3 chemokines. The production of CXCR3 ligands via Fas ligation is responsible for recruiting activated lymphocytes and can further activate innate immune cells as well as augment the production of effector cytokines such as IFN-{gamma}. As a consequence of this process, the amplification of liver inflammation can occur by recruitment of more lymphocytes to the liver compartment. Through Fas/FasL-mediated signaling, these liver-infiltrating lymphocytes induce hepatocyte death and the production of CXCR3 ligands. Such an effect is able to cause sustained liver damage, especially in the presence of a chronic HCV infection. Using inhibitors of the Fas pathway or the CXCR3 systems would provide better treatment for liver disease and may reduce the incidence of liver fibrosis in HCV infection.


   Acknowledgments
 
We thank the other members of the Young Hahn laboratory who assisted in the collection and analysis of these data, specifically, Susan Landes and Barbara Small.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by Grants DK063222 (to Y.S.H.) and Training Fellowship 5T32AI10749608 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Young S. Hahn, Department of Microbiology and Beirne Carter Center, University of Virginia, HSC Box 801386, Charlottesville, VA 22908. E-mail address: ysh5e{at}virginia.edu Back

3 Abbreviations used in this paper: HCV, hepatitis C virus; FasL, Fas ligand; IP-10, IFN-inducible protein 10; Mig, monokine induced by IFN-{gamma}; ITAC, IFN-inducible T cell {alpha} chemoattractant; IHC, immunohistochemistry; FFPE, Formalin-fixed paraffin-embedded; RPA, RNase protection assay; ALT, alanine aminotransferase. Back

Received for publication November 3, 2005. Accepted for publication February 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. He, X. S., B. Rehermann, F. X. Lopez-Labrador, J. Boisvert, R. Cheung, J. Mumm, H. Wedemeyer, M. Berenguer, T. L. Wright, M. M. Davis, H. B. Greenberg. 1999. Quantitative analysis of hepatitis C virus-specific CD8+ T cells in peripheral blood and liver using peptide-MHC tetramers. Proc. Natl. Acad. Sci. USA 96: 5692-5697. [Abstract/Free Full Text]
  2. Crispe, I. N.. 2003. Hepatic T cells and liver tolerance. Nat. Rev. Immunol. 3: 51-62. [Medline]
  3. John, B., I. N. Crispe. 2004. Passive and active mechanisms trap activated CD8+ T cells in the liver. J. Immunol. 172: 5222-5229. [Abstract/Free Full Text]
  4. Janssen, O., J. Qian, A. Linkermann, D. Kabelitz. 2003. CD95 ligand–death factor and costimulatory molecule?. Cell Death Differ. 10: 1215-1225. [Medline]
  5. Alderson, M. R., R. J. Armitage, E. Maraskovsky, T. W. Tough, E. Roux, K. Schooley, F. Ramsdell, D. H. Lynch. 1993. Fas transduces activation signals in normal human T lymphocytes. J. Exp. Med. 178: 2231-2235. [Abstract/Free Full Text]
  6. Sakata, K., A. Sakata, N. Vela-Roch, R. Espinosa, A. Escalante, L. Kong, T. Nakabayashi, J. Cheng, N. Talal, H. Dang. 1998. Fas (CD95)-transduced signal preferentially stimulates lupus peripheral T lymphocytes. Eur. J. Immunol. 28: 2648-2660. [Medline]
  7. Pimentel-Muinos, F. X., B. Seed. 1999. Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity 11: 783-793. [Medline]
  8. Desbarats, J., M. K. Newell. 2000. Fas engagement accelerates liver regeneration after partial hepatectomy. Nat. Med. 6: 920-923. [Medline]
  9. Neff, T. A., R. F. Guo, S. B. Neff, J. V. Sarma, C. L. Speyer, H. Gao, K. D. Bernacki, M. Huber-Lang, S. McGuire, L. M. Hoesel, et al 2005. Relationship of acute lung inflammatory injury to Fas/FasL system. Am. J. Pathol. 166: 685-694. [Abstract/Free Full Text]
  10. Garcia-Lopez, M. A., F. Sanchez-Madrid, J. M. Rodriguez-Frade, M. Mellado, A. Acevedo, M. I. Garcia, J. P. Albar, C. Martinez, M. Marazuela. 2001. CXCR3 chemokine receptor distribution in normal and inflamed tissues: expression on activated lymphocytes, endothelial cells, and dendritic cells. Lab. Invest. 81: 409-418. [Medline]
  11. Farber, J. M., M. Bernhard. 2003. CXCR3. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 2003-2008. Academic Press, London.
  12. Luster, A. D.. 2003. MIG. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 1111-1118. Academic Press, London.
  13. Farber, J. M.. 2003. IP-10. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 1103-1110. Academic Press, London.
  14. Blanpain, C. P., M. Parmentier. 2003. CCR5. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 2067-2080. Academic Press, London.
  15. Bernhard, M.. 2003. CXCR4. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 2009-2018. Academic Press, London.
  16. Mackay, C. R.. 2003. CCR3. J. Oppenheim, and M. Feldmann, eds. Cytokine Reference 2055-2062. Academic Press, London.
  17. Wang, J. T., J. C. Sheu, J. T. Lin, T. H. Wang, D. S. Chen. 1992. Detection of replicative form of hepatitis C virus RNA in peripheral blood mononuclear cells. J. Infect. Dis. 166: 1167-1169. [Medline]
  18. Muller, H., E. Pfaff, T. Gocser, B. Kallinoski, C. Solbach, L. Theilmann. 1993. Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication. J. Gen. Virol. 74: 669-676. [Abstract/Free Full Text]
  19. Bronowicki, J. P., M. A. Loriot, V. Thiers, Y. Grignon, A. L. Zignego, C. Brechot. 1998. Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice. Hepatology 28: 211-218. [Medline]
  20. Soguero, C., M. Joo, K. A. Chianese-Bullock, D. T. Nguyen, K. Tung, Y. S. Hahn. 2002. Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model. J. Virol. 76: 9345-9354. [Abstract/Free Full Text]
  21. Cruise, M. W., H. M. Melief, J. Lukens, C. Soguero, Y. S. Hahn. 2005. Increased Fas ligand expression of CD4+ T cells by HCV core induces T cell-dependent hepatic inflammation. J. Leukocyte Biol. 78: 412-425. [Abstract/Free Full Text]
  22. Belperio, J. A., M. P. Keane, M. D. Burdick, J. P. Lynch, III, Y. Y. Xue, K. Li, D. J. Ross, R. M. Strieter. 2002. Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome. J. Immunol. 169: 1037-1049. [Abstract/Free Full Text]
  23. Kennedy, N. J., J. Q. Russell, N. Michail, R. C. Budd. 2001. Liver damage by infiltrating CD8+ T cells is Fas dependent. J. Immunol. 167: 6654-6662. [Abstract/Free Full Text]
  24. Schaub, F. J., W. C. Liles, N. Ferri, K. Sayson, R. A. Seifert, D. F. Bowen-Pope. 2003. Fas and Fas-associated death domain protein regulate monocyte chemoattractant protein-1 expression by human smooth muscle cells through caspase- and calpain-dependent release of interleukin-1{alpha}. Circ. Res. 93: 515-522. [Abstract/Free Full Text]
  25. Marra, F., R. G. Romanelli, C. Giannini, P. Failli, S. Pastacaldi, M. C. Arrighi, M. Pinzani, G. Laffi, P. Montalto, P. Gentilini. 1999. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells. Hepatology 29: 140-148. [Medline]
  26. Guo, Z., M. Zhang, H. Tang, X. Cao. 2005. Fas signal links innate and adaptive immunity by promoting dendritic-cell secretion of CC and CXC chemokines. Blood 106: 2033-2041. [Abstract/Free Full Text]
  27. Suzuki, I., P. J. Fink. 2000. The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells. Proc. Natl. Acad. Sci. USA 97: 1707-1712. [Abstract/Free Full Text]
  28. Park, D. R., A. R. Thomsen, C. W. Frevert, U. Pham, S. J. Skerrett, P. A. Kiener, W. C. Liles. 2003. Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages. J. Immunol. 170: 6209-6216. [Abstract/Free Full Text]
  29. Ma, Y., H. Liu, H. Tu-Rapp, H. J. Thiesen, S. M. Ibrahim, S. M. Cole, R. M. Pope. 2004. Fas ligation on macrophages enhances IL-1R1-Toll-like receptor 4 signaling and promotes chronic inflammation. Nat. Immunol. 5: 380-387. [Medline]
  30. Shimada, M., A. Andoh, Y. Araki, Y. Fujiyama, T. Bamba. 2001. Ligation of the Fas antigen stimulates chemokine secretion in pancreatic cancer cell line PANC-1. J. Gastroenterol. Hepatol. 16: 1060-1067. [Medline]
  31. Dosreis, G. A., V. M. Borges, W. A. Zin. 2004. The central role of Fas-ligand cell signaling in inflammatory lung diseases. J. Cell. Mol. Med. 8: 285-293. [Medline]
  32. Rotondi, M., E. Lazzeri, P. Romagnani, M. Serio. 2003. Role for interferon-{gamma} inducible chemokines in endocrine autoimmunity: an expanding field. J. Endocrinol. Invest. 26: 177-180. [Medline]
  33. Gerard, C., B. J. Rollins. 2001. Chemokines and disease. Nat. Immunol. 2: 108-115. [Medline]
  34. Bieche, I., T. Asselah, I. Laurendeau, D. Vidaud, C. Degot, V. Paradis, P. Bedossa, D. C. Valla, P. Marcellin, M. Vidaud. 2005. Molecular profiling of early stage liver fibrosis in patients with chronic hepatitis C virus infection. Virology 332: 130-144. [Medline]
  35. Harvey, C. E., J. J. Post, P. Palladinetti, A. J. Freeman, R. A. Ffrench, R. K. Kumar, G. Marinos, A. R. Lloyd. 2003. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J. Leukocyte Biol. 74: 360-369. [Abstract/Free Full Text]
  36. Helbig, K. J., A. Ruszkiewicz, L. Semendric, H. A. Harley, S. R. McColl, M. R. Beard. 2004. Expression of the CXCR3 ligand I-TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation. Hepatology 39: 1220-1229. [Medline]
  37. Itoh, Y., A. Morita, K. Nishioji, S. Narumi, T. Toyama, Y. Daimon, H. Nakamura, T. Kirishima, T. Okanoue. 2001. Clinical significance of elevated serum interferon-inducible protein-10 levels in hepatitis C virus carriers with persistently normal serum transaminase levels. J. Viral Hepat. 8: 341-348. [Medline]
  38. Butera, D., S. Marukian, A. E. Iwamaye, E. Hembrador, T. J. Chambers, A. M. Di Bisceglie, E. D. Charles, A. H. Talal, I. M. Jacobson, C. M. Rice, L. B. Dustin. 2005. Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C. Blood 106: 1175-1182. [Abstract/Free Full Text]
  39. Xanthou, G., T. J. Williams, J. E. Pease. 2003. Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation. Eur. J. Immunol. 33: 2927-2936. [Medline]
  40. Heise, C. E., A. Pahuja, S. C. Hudson, M. S. Mistry, A. L. Putnam, M. M. Gross, P. A. Gottlieb, W. S. Wade, M. Kiankarimi, D. Schwarz, et al 2005. Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist. J. Pharmacol. Exp. Ther. 313: 1263-1271. [Abstract/Free Full Text]
  41. Itoh, Y., A. Morita, K. Nishioji, H. Fujii, H. Nakamura, T. Kirishima, T. Toyama, N. Yamauchi, Y. Nagao, S. Narumi, T. Okanoue. 2001. Time course profile and cell-type-specific production of monokine induced by interferon-{gamma} in Concanavalin A-induced hepatic injury in mice: comparative study with interferon-inducible protein-10. Scand. J. Gastroenterol. 36: 1344-1351. [Medline]
  42. Romagnani, P., F. Annunziato, L. Lasagni, E. Lazzeri, C. Beltrame, M. Francalanci, M. Uguccioni, G. Galli, L. Cosmi, L. Maurenzig, et al 2001. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J. Clin. Invest. 107: 53-63. [Medline]
  43. Bonacchi, A., P. Romagnani, R. G. Romanelli, E. Efsen, F. Annunziato, L. Lasagni, M. Francalanci, M. Serio, G. Laffi, M. Pinzani, et al 2001. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J. Biol. Chem. 276: 9945-9954. [Abstract/Free Full Text]
  44. Apolinario, A., P. L. Majano, R. Lorente, O. Nunez, G. Clemente, C. Garcia-Monzon. 2005. Gene expression profile of T-cell-specific chemokines in human hepatocyte-derived cells: evidence for a synergistic inducer effect of cytokines and hepatitis C virus proteins. J. Viral Hepat. 12: 27-37. [Medline]
  45. Machida, K., K. Tsukiyama-Kohara, E. Seike, S. Tone, F. Shibasaki, M. Shimizu, H. Takahashi, Y. Hayashi, N. Funata, C. Taya, et al 2001. Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins. J. Biol. Chem. 276: 12140-12146. [Abstract/Free Full Text]
  46. Ray, R. B., K. Meyer, R. Ray. 1996. Suppression of apoptotic cell death by hepatitis C virus core protein. Virology 226: 176-182. [Medline]
  47. Gremion, C., B. Grabscheid, B. Wolk, D. Moradpour, J. Reichen, W. Pichler, A. Cerny. 2004. Cytotoxic T lymphocytes derived from patients with chronic hepatitis C virus infection kill bystander cells via Fas-FasL interaction. J. Virol. 78: 2152-2157. [Abstract/Free Full Text]
  48. Mahalingam, S., J. M. Farber, G. Karupiah. 1999. The interferon-inducible chemokines MuMig and Crg-2 exhibit antiviral activity in vivo. J. Virol. 73: 1479-1491. [Abstract/Free Full Text]
  49. Mihm, S., S. Schweyer, G. Ramadori. 2003. Expression of the chemokine IP-10 correlates with the accumulation of hepatic IFN-{gamma} and IL-18 mRNA in chronic hepatitis C but not in hepatitis B. J. Med. Virol. 70: 562-570. [Medline]
  50. Ahmad, A., F. Alvarez. 2004. Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis. J. Leukocyte Biol. 76: 743-759. [Abstract/Free Full Text]
  51. Whiting, D., G. Hsieh, J. J. Yun, A. Banerji, W. Yao, M. C. Fishbein, J. Belperio, R. M. Strieter, B. Bonavida, A. Ardehali. 2004. Chemokine monokine induced by IFN-{gamma}/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production. J. Immunol. 172: 7417-7424. [Abstract/Free Full Text]
  52. Dufour, J. H., M. Dziejman, M. T. Liu, J. H. Leung, T. E. Lane, A. D. Luster. 2002. IFN-{gamma}-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J. Immunol. 168: 3195-3204. [Abstract/Free Full Text]
  53. Eichhorst, S. T., A. Krueger, S. Muerkoster, S. C. Fas, A. Golks, U. Gruetzner, L. Schubert, C. Opelz, M. Bilzer, A. L. Gerbes, P. H. Krammer. 2004. Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice. Nat. Med. 10: 602-609. [Medline]
  54. Doggrell, S. A.. 2004. Suramin: potential in acute liver failure. Expert Opin. Investig. Drugs 13: 1361-1363. [Medline]
  55. Yang, Y. F., T. Mukai, P. Gao, N. Yamaguchi, S. Ono, H. Iwaki, S. Obika, T. Imanishi, T. Tsujimura, T. Hamaoka, H. Fujiwara. 2002. A non-peptide CCR5 antagonist inhibits collagen-induced arthritis by modulating T cell migration without affecting anti-collagen T cell responses. Eur. J. Immunol. 32: 2124-2132. [Medline]
  56. Ondeykal, J. G., K. B. Herath, H. Jayasuriya, J. D. Polishook, G. F. Bills, A. W. Dombrowski, M. Mojena, G. Koch, J. DiSalvo, J. DeMartino, et al 2005. Discovery of structurally diverse natural product antagonists of chemokine receptor CXCR3. Mol. Divers. 9: 123-129. [Medline]




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