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Originally published online as doi:10.1189/jlb.0303093 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:360-369.)
© 2003 by Society for Leukocyte Biology

Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation

Charles E. Harvey*, Jeffrey J. Post{dagger}, Patricia Palladinetti{dagger}, Anthony J. Freeman*, Rosemary A. Ffrench{ddagger}, Rakesh K. Kumar{dagger}, George Marinos* and Andrew R. Lloyd{dagger},1

* Viral Hepatitis Research Unit, Department of Gastroenterology, Prince of Wales Hospital, Sydney, New South Wales, Australia; and
{dagger} Inflammation Research Unit, Department of Pathology, and
{ddagger} Department of Immunology and Infectious Diseases, Sydney Children’s Hospital, and the School of Women’s and Children’s Health, University of New South Wales, Sydney, Australia

1Correspondence: University of NSW, Inflammation Research Unit, School of Medical Sciences, Sydney, NSW 2052, Australia. E-mail: A.lloyd{at}unsw.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The factors influencing lymphocyte trafficking to the liver lobule during chronic hepaititis C virus (HCV) infection are currently not well defined. Interferon-{gamma}-inducible protein 10 (IP-10), a chemokine that recruits activated T lymphocytes, has recently been shown by in situ hybridization to be expressed in the liver during chronic HCV infection. This study sought to define the cellular source of IP-10 in the liver by immunohistochemistry, to examine the expression of its receptor, CXCR3, on T lymphocytes isolated from blood and liver tissue, and to correlate IP-10 expression with the histological markers of inflammation and fibrosis. IP-10 was expressed by hepatocytes but not by other cell types within the liver, and the most intense immunoreactivity was evident in the areas of lobular inflammation. The IP-10 receptor was expressed on a significantly higher proportion of T lymphocytes in the liver compared with blood. CD8 T lymphocytes, which predominate in the liver lobule, were almost uniformly CXCR3-positive. The expression of IP-10 mRNA correlated with lobular necroinflammatory activity but not with inflammation or fibrosis in the portal tracts. These findings suggest that IP-10 may be induced by HCV within hepatocytes and may be important in the pathogenesis of chronic HCV infection, as recruitment of inflammatory cells into the lobule is an important predictor of disease progression.

Key Words: pathogenesis • liver • trafficking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) infection is the leading cause of liver disease-related morbidity and mortality worldwide [1 ]. Histological analysis of liver biopsy specimens in acute and chronic hepatitis C typically demonstrates infiltration of the liver by mononuclear cells, including T and B lymphocytes, and natural killer (NK) cells [2 , 3 ]. Notably, portal lymphoid aggregates and lobular inflammation of varying severity are more likely to be seen in hepatitis C than hepatitis A or B [4 5 6 ]. Long-term infection with HCV is associated with progressive infiltration of the liver parenchyma by mononuclear cells, fibrosis, cirrhosis, and the risk of developing hepatocellular carcinoma [7 ]. The mechanisms responsible for liver injury during chronic HCV infection are not well understood, but increasing evidence suggests that the host immune response plays a critical role in HCV pathogenesis [8 , 9 ]. In particular, lysis of HCV-infected liver cells by virus-specific cytotoxic T lymphocytes, in the absence of viral clearance, may cause liver cell damage [10 , 11 ]. The factors regulating recruitment of CD8 T lymphocytes and other cellular components of the host response to the liver during HCV infection remain to be elucidated. However, chemokines are likely candidates for such activity.

Chemokines are small (8–10 kDa), specialized, proinflammatory cytokines best known for their ability to recruit and activate specific subsets of leukocytes at sites of inflammation or an immune response. The presence of conserved cysteine residues near the N terminus allows chemokines to be divided into four structurally related subfamilies (CC, CXC, CX3C, C; reviewed in detail elsewhere [12 13 14 ]). Expression of chemokines is observed in many disease states, suggesting that they play an important role in the pathogenesis of a wide range of infective, inflammatory, and autoimmune diseases. The ability to prevent or ameliorate the severity of infective and allergic diseases by inhibition of the activity of specific chemokines further supports this hypothesis [15 , 16 ].

Recent studies have reported the expression of interferon-{gamma} (IFN-{gamma})-inducible protein-10 (IP-10), a CXC chemokine that is chemotactic for activated T lymphocytes and NK cells, in the liver of patients with chronic HCV infection [17 18 19 ]. However, the cellular source of IP-10 identified in these reports is conflicting. This study sought to define the cellular source of IP-10 in the liver by immunohistochemistry, to examine the expression of its receptor CXCR3 on T lymphocytes isolated from blood and liver tissue, and to correlate IP-10 expression with the histological markers of inflammation and fibrosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue samples and patient characteristics
Liver biopsy tissue was obtained from 33 patients with stable chronic hepatitis C attending the Liver Clinic at the Prince of Wales Hospital in Sydney, Australia, for assessment and treatment. A portion of the liver biopsy tissue was formalin-fixed and paraffin-embedded for histological examination and immunohistochemistry (n=22). If sufficient material were available, another portion was snap-frozen in liquid nitrogen in a RNase-free vial (NUNC Inc., Naperville, IL) for subsequent RNA extraction (n=6) or collected into sterile saline for subsequent flow cytometry analysis to assess chemokine receptor expression on liver-infiltrating T lymphocytes (n=5). At the time of liver biopsy, serum was collected from the patients for biochemical and virological analysis. The mean age of the patients was 38.5 years (range, 21–70). All were anti-HCV antibody-positive by third-generation enzyme-linked immunosorbent assay and had detectable serum HCV RNA by polymerase chain reaction. All patients had histologically proven chronic hepatitis, and all but one had elevated alanine-aminotransferase (ALT) levels (mean, 111.9±63.3 IU/L; range, 36–275; normal range, 17–63). HCV viral load was determined on 17 patients using the Amplicor Monitor assay, according to the manufacturer’s instructions (Roche Diagnostic Systems, Nutley, NJ; mean, 2.14x106±2.07x106 copies/ml; range, 3.2x105–9.1x106). All patients were hepatitis B surface antigen-negative, and other causes of chronic liver disease had been excluded by clinical and laboratory assessments. The 33 patients in this report were selected before the immunohistochemical studies to represent a range of histological scores on liver biopsy. Control liver tissue was obtained from macroscopically normal areas of tissue in specimens resected for metastatic colonic carcinoma. Tonsillar tissue was obtained from routine tonsillectomies performed at the Prince of Wales Hospital. All specimens were collected after written informed consent was obtained. The local institutional human research ethics committee approved the study.

Histological assessment
An experienced histopathologist evaluated all patient and control liver tissues for features of necroinflammatory activity and fibrosis. These features were scored in all samples using the Scheuer index [20 ]. The histopathologist was blinded to the outcome of other analyses.

RNase protection assay
Quantitative analysis of chemokine mRNA expression in HCV-infected liver was determined by a RNase protection assay, which was performed according to the manufacturer’s instructions using a multiprobe chemokine template set (PharMingen, San Diego, CA). Briefly, liver tissue was collected at the time of biopsy, transferred immediately to a RNase-free cryovial, and snap-frozen in liquid nitrogen. Frozen tissue was homogenized in the presence of TRI-Reagent (Sigma Chemical Co., St. Louis, MO) using diethylpyrocarbonate-treated, autoclaved, disposable Eppendorf homogenizers, and total RNA was extracted according to the manufacturer’s protocol. RNA concentrations and purity were determined by spectrophotometry. Template DNA was used to generate [{gamma}32P] uridine 5'-triphosphate (3000 Ci/mmol, 10 mCi/ml; Amersham, Piscataway, NJ)-radiolabeled probes in the presence of a dNTP pool using a T7 RNA polymerase. The template set contained probes for the CXC chemokines IP-10 and interleukin-8 and the C–C chemokines regulated on activation normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, monocyte chemoattractant protein-1, and I-309. Hybridization with 1–3 µg each target RNA was performed overnight followed by digestion with DNase-free RNase. The samples were treated with proteinase K, extracted with phenol/chloroform, and precipitated in the presence of ammonium acetate. The samples were loaded on an acrylamide-urea sequencing gel next to the labeled probes and were run at 40 W with 0.5% Tris borate/EDTA. The gel was absorbed to filter paper, dried under vacuum, and exposed on Kodak X-AR film with an intensifying screen at -70°C. IP-10 mRNA expression was quantified by densitometric analysis of the autoradiograph, and data are presented as densitometry units (dU) after normalization, according to the reference glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal in each lane. RNA extracted from explant liver tissue was used as a control.

Flow cytometry
Four-color flow cytometry was performed to assess cell phenotype, activation status, and chemokine receptor expression on T lymphocyte subsets in paired blood and liver samples obtained from five patients with chronic HCV infection. Peripheral blood mononuclear cells were isolated from whole blood by Ficoll-Hypaque density-gradient centrifugation, washed twice in sterile phosphate-buffered saline (PBS), and resuspended in preparation for staining at 1 x 106 cells/mL in PBS. Liver-infiltrating mononuclear cells (LIMC) were obtained by physical disaggregation of the liver tissue. LIMC were washed twice in PBS and resuspended at 1 x 106 cell/ml in preparation for staining. Antibodies used in this study included anti-CD3 APC, anti-CD4 [peridinin chlorophyll protein (PerCP)], anti-CD8 [phycoerythrin (PE)], anti-CD69 (PE; BD PharMingen, San Diego, CA), and anti-CXCR3 [fluorescein isothiocyanate (FITC); R&D Systems, Minneapolis, MN] for 30 min at 4°C and were washed twice in PBS and resuspended in 500 µL 2% paraformaldehyde. Analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Lymphocytes were gated on the basis of forward- and side-scatter profiles, and the results were analyzed using Cell Quest Pro software (Becton Dickinson). The positioning of analysis regions for blood and liver samples was determined against a panel of isotype-matched, negative-control antibodies conjugated to FITC, PE, PerCP, or APC. The region position was set such that at least 99% of cells in negative-control tubes were excluded.

Immunohistochemistry
Immunohistochemistry was performed to assess chemokine and leukocyte surface-marker expression in the liver of 22 patients. Liver tissue was fixed in 4% buffered formaldehyde and embedded in paraffin. Sections 4-µm thick were mounted on slides coated with 3-aminopropyl-triethoxy-silane (Sigma Chemical Co.) and dried for 12 h at 37°C. Sections were deparaffinized in xylene and rehydrated in a series of graded ethanol and Tris-buffered saline (TBS) solutions. Antigen retrieval was performed by microwave heating for 15 min at 750 W in citrate buffer (pH 6.0). Sections were allowed to cool in the same buffer, washed in TBS, and blocked for 15 min at room temperature in TBS containing 10% preimmune serum from the species in which the secondary antibody was raised. Chemokine expression was also determined in fresh frozen liver tissue. Cryostat sections were cut 4-µm thick and fixed briefly in acetone for 10 min. Sections were blocked as above and then incubated with primary antibody. Primary antibodies were incubated on the sections overnight at 4°C at the following concentrations: goat polyclonal anti-human IP-10 antibody (R&D Systems), 5 µg/ml; rabbit polyclonal anti-human CD3 antibody (DAKO Corp., Carpinteria, CA), 1:100 dilution; mouse anti-human CD20 and CD68 antibodies (DAKO Corp.), 1:200 dilution. All antibodies were diluted in TBS–2% bovine serum albumin. Negative-control antibodies consisted of species-matched and where appropriate, immunoglobulin G (IgG) subclass-matched Ig fractions, used at the same dilution as the primary antibodies. Inflamed tonsil sections were used as positive-control tissue for IP-10, CD3, CD20, and CD68 staining. Bound IP-10 antibody was detected with a mouse antigoat alkaline phosphatase-conjugated antibody (Sigma Chemical Co.) used at 1:200 dilution for 30 min at room temperature and visualized by the addition of FastRed (Sigma Chemical Co.). Bound murine and rabbit antibodies were detected with goat anti-mouse biotin-conjugated or goat anti-rabbit biotin-conjugated secondary antibodies, respectively (DAKO Corp.), used at 1:200 dilution, followed by streptavidin-horseradish peroxidase conjugate (Sigma Chemical Co.; 1:200 dilution). Incubations were performed for 30 min at room temperature. The enzyme complex was visualized by the addition of 3',3'-diaminobenzidine tetrachloride (Sigma Chemical Co.). Sections were washed with TBS between incubations.

Specificity of IP-10 immunostaining
Blocking experiments were performed to confirm the specificity of IP-10 immunostaining. Goat anti-human IP-10 antibody (5 µg/ml) was left untreated or preadsorbed with 100 ng/ml recombinant human IP-10 (rhIP-10; R&D Systems), a concentration of recombinant protein calculated to be limiting in relation to antibody, or with 500 ng/ml rhIP-10, a protein concentration calculated to be in excess of antibody, for 1 h at 37°C in diluent buffer containing TBS/10% normal mouse serum/0.1% Triton X-100. The untreated or preabsorbed IP-10 antibody was used as the primary antibody and incubated on HCV-infected liver, tonsil (positive-control tissue), and normal liver (negative-control tissue) sections as described above.

Morphometry
Morphometric analysis of the sections was performed to quantify T lymphocytes (CD3+), B lymphocytes (CD20+), and macrophages (CD68+) in HCV-infected liver. Morphometry was performed as described previously with modifications [21 ]. Briefly, a systematic sampling procedure was used with a random start position on the slide [22 ]. Stained sections were examined with an Olympus microscope to which a video camera was attached, and images were projected on a color monitor. The screen was overlaid with a coherent point-counting lattice of 8 x 5 points, spaced at 5-cm apart, of which one point was designated as the test point. To examine each slide, the test grid was initially located at one corner of the section. A random start was achieved by using a computer-generated random number to select one of the 40 points and moving the stage so that the tissue underlying the selected point was superimposed on the location marked by the test point. Thereafter, fields were systematically sampled at 1-mm intervals using the Vernier stage movement. Cell counts and area calculations were divided into portal and lobular fields. Two independent observers (C. E. H. and J. J. P.), masked to the histological score of the section, simultaneously examined every field. On average, 18 fields were counted per section.

Statistical analysis
The nonparametric Spearman correlation was used for all correlations. The means of paired samples were compared using a paired Student’s t-test. All statistical analyses were performed using GraphPad Prism v2.01 (GraphPad Software Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chemokine mRNA expression
Chemokine mRNA expression was examined in liver by RNase protection assay. IP-10, RANTES, MIP-1ß, and I-309 were all found to be up-regulated in HCV-infected subjects compared with uninfected control tissue (Fig. 1A ). In particular, IP-10 mRNA expression was induced up to 30-fold higher in HCV-infected tissue compared with control liver tissue and was more abundantly expressed in comparison with the other chemokines analyzed.



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  		Figure 1. (A). IP-10, RANTES, MIP-1ß, and I-309 mRNA expression in HCV-infected subjects was detected by RNase protection assay as described in Materials and Methods. The results were analyzed by densitometry, normalized against GAPDH expression, and presented in dU. Chemokine expression in liver of HCV-infected subjects is presented in shaded bars and control liver, in solid bars. IP-10 was detected in liver of 6/6 HCV-infected subjects and ranged between 368 and 3504 dU (median, 899.5 dU). RANTES expression was detected in liver of 5/6 HCV subjects and ranged between 170 and 471 dU (median, 199 dU). MIP-1ß expression was detected in 3/6 HCV subjects and ranged between 33 and 217 dU (median, 61 dU). I-309 expression was detected in liver of 6/6 HCV subjects and ranged between 43 and 190 dU (median, 108 dU). Only IP-10 and I-309 were detected in control liver (IP-10 range, 54–120, median, 87 dU; I-309 range, 21–27, median 24 dU). (B) IP-10 mRNA expression was determined and correlated with the Scheuer score of histological assessment. A positive relationship was observed (r=0.88; P<0.033).

 
IP-10 expression was detected in all HCV-infected liver samples and ranged between 368 and 3504 dU (median, 899.5 dU), whereas control liver tissue expressed very little IP-10 (54–120 dU; median, 87 dU). IP-10 expression strongly correlated with the total Scheuer score (r=0.88; P=0.033; Fig. 1B ) and more specifically, with the lobular inflammation component of the score (r=0.878; P<0.033) but not with the portal inflammation (r=0.65; P>0.1) or fibrosis scores (r=0.61; P>0.1; Table 1 ).


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  		Table 1. Correlation of Cell Type and Chemokine mRNA Expression with Components of the Scheuer Score

 
RANTES expression was detected in 5/6 HCV-infected samples and ranged between 170 and 471 dU (median, 199 dU; Fig. 1A ). Low-level MIP-1ß expression was detected in 3/6 HCV-infected subjects and ranged between 33 and 217 dU (median, 61 dU; Fig. 1A ). RANTES and MIP-1ß were not detected in uninfected control liver. I-309 expression was detected in all samples tested. I-309 expression ranged between 43 and 190 dU (median, 108 dU) in HCV-infected liver and 21 and 27 dU (median, 24 dU) in control liver (Fig. 1A) . The expression of RANTES, MIP-1ß, and I-309 did not correlate with the Scheuer score or its components.

Leukocyte infiltration in chronic HCV
The expression of cell-surface markers (CD3, CD20, and CD68) in the liver of patients with a range of histological scores was determined by immunohistochemistry. CD3+ T cells were evident in the periportal region and throughout the liver lobule (Fig. 2A ). T cell numbers in the lobule detected by immunohistochemistry correlated with the histopathologist’s assessment of lobule inflammatory scores (Spearman r=0.71, P<0.0002; Table 1 ). No such correlation was evident for portal T cell numbers and the portal inflammation score (r=0.41, P>0.05), suggesting that other leukocytes (e.g., B cells, see below) predominate in this site. There was no correlation between T cell numbers in the liver and age, sex, ALT, or viral load (data not shown). Total T cell numbers (combined portal and lobular T cell counts) correlated with the total Scheuer score (r=0.74, P<0.0001), lobular inflammation (r=0.77, P<0.0001), portal inflammation (r=0.55, P<0.01), and fibrosis scores (r=0.54, P<0.01; Table 1 ).



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  		Figure 2. Immunoperoxidase staining of HCV-infected liver with (A) anti-CD3, (B) anti-CD20 (original magnification, x400), and (C) anti-CD68 (original magnification, x200).

 
Consistent with the findings above, CD20+ B cells were seen in and adjacent to portal tracts and only occasionally in the liver lobule, indicating that the majority of the lymphocytes in the lobule were T cells (Fig. 2B) . B cells associated with portal tracts were often present in aggregations with the appearance of germinal centers. The numbers of lobule-associated B cells did not correlate with the lobular inflammation score (r=0.39, P>0.05), nor did B cell numbers in portal areas exhibit significant correlation with portal inflammation. Combined B cell counts correlated weakly with the total Scheuer score (r=0.45, P<0.05; Table 1 ).

CD68+ macrophages were scattered at relatively lower frequency throughout the liver sections (Fig. 2C) and did not correlate with any component of the histology score or patient characteristics (Table 1) .

Flow cytometry
Expression of the chemokine receptor CXCR3 on T lymphocytes isolated from paired blood and liver samples from patients with chronic HCV infection was examined by four-color flow cytometry.

The percentage of T lymphocytes found to express CXCR3 in liver samples was consistently and significantly higher than T lymphocytes in paired blood samples (liver mean, 94.67%; blood, 58.97%; P=0.0061; Fig. 3A and 3C ). CXCR3 was found expressed on CD4 and CD8 T lymphocytes (Fig. 3A) . The percentage of CD8-positive T lymphocytes expressing CXCR3 in the blood was consistently higher than the percentage of CD4-positive T lymphocytes (mean CD8, 70.98%; CD4, 54.92%; Fig. 3D ). In the liver, however, the reverse was observed. CD4-positive T lymphocytes were consistently more likely to express CXCR3 than CD8-positive T lymphocytes; however, the observed difference was small and not statistically different (mean CD8, 91.8%; CD4, 94.7%; Fig. 3D ). Analysis of CD69 expression, a marker of recent activation, revealed the majority of CD3+ T lymphocytes in the liver expresses CD69 (80%) compared with just 6% in the blood (Fig. 3B) . The expression of CXCR3 on CD69+ and CD69- T cell subsets in blood and liver was also examined. In the liver, the majority of CD69+cells were CXCR3-positive (90%) as were a significant proportion of CD69- cells (47%). In the blood, CXCR3 was expressed on a smaller percentage of CD69+ T cells compared with liver (35%) and on only a minority of CD69- cells (19%).



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  		Figure 3. (A) Analysis of CXCR3 expression on total T lymphocytes (CD3+) and on T lymphocyte subsets (CD3/CD4+ or CD3/CD8+) isolated from paired liver and blood samples obtained from a subject with chronic HCV. Cells were stained with APC-conjugated anti-CD3, PerCP-conjugated anti-CD4, PE-conjugated anti-CD8, and FITC-conjugated anti-CXCR3. Isotype-matched mouse Igs conjugated to each of the fluorochromes were used as negative controls. The analysis region was set according to the staining of negative-control antibodies such that no more than 1% of cells were positive (as shown, lower two panels). Percentages refer to the cells in the marked regions. The results are representative of those obtained from five different subjects. (B) Analysis of CD69 expression on T lymphocytes isolated from paired blood and liver samples obtained from a subject with chronic HCV infection. The expression of CXCR3 on CD69+ and CD69- subpopulations of CD3+ T lymphocytes is also shown. Percentages refer to the cells in the marked regions. Regions were set as above. The results are representative of those obtained from five different subjects. (C) CXCR3 expression was analyzed on CD3 T lymphocytes in paired blood and liver samples (n=5), and the percentage of positive cells in each compartment was graphed. Mean percentage of CXCR3 T lymphocytes in blood = 58.9% [SD±19.5; 95% confidence interval (CI), 38.4–79.5]; mean percentage of CXCR3 T lymphocytes in liver = 94.6% (SD±2.3; 95% CI, 92.2–97.1). The means are statistically different (P=0.0061). (D) The expression of CXCR3 on CD4 and CD8 T lymphocytes in blood and liver was determined (n=5), and the mean percent-positive was graphed. Standard deviation error bars are shown. Blood: Mean % CD4 = 54.9 (SD±21.0; 95% CI, 32.8–77.0); CD8 = 70.9 (SD±25.4; 95% CI, 39.3–102.6). Liver: Mean % CD4 = 94.7 (SD±2.0; 95% CI, 92.5–96.8); CD8 = 91.8 (SD±6.1; 95% CI, 84.1–99.5).

 
Cellular localization of IP-10
The expression of IP-10 protein and cellular localization was determined by immunohistochemistry. The analysis revealed strong IP-10 staining in the cytoplasm of hepatocytes but not in other cell types. Staining was detected in hepatocytes throughout the sections, and the most intense staining was observed near areas of lobular inflammation (Fig. 4A 4B 4C ). All sections demonstrated some degree of IP-10 staining. The same pattern of IP-10 staining was observed in fresh frozen tissue (data not shown) and formalin-fixed paraffin-embedded samples.



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  		Figure 4. Immuno-alkaline phosphatase/FastRed staining for IP-10 in chronic HCV-infected livers with a range of histological scores. In chronic HCV, IP-10 is strongly detected in the cytoplasm of hepatocytes (positive cells stain red; B and C). Notably, there was no evidence of IP-10 expression by other populations of cells within the liver. The intensity of IP-10 expression appears to increase with the severity of histological disease. (A) Little IP-10 expression. Scheuer score, 1; lobular inflammation score, 1. (B) Moderate IP-10 expression. Scheuer score, 4; lobular inflammation score, 2. (C) Intense IP-10 expression. Scheuer score, 9; lobular inflammation score, 3. (Original magnification, x400.)

 
Specificity of IP-10 immunostaining
Preadsorption of the anti-IP-10 antibody with rhIP-10 specifically blocked IP-10 staining in HCV-infected liver in a concentration-dependent manner. Adsorption of 5 µg/ml anti-IP-10 antibody with 100 ng/ml rhIP-10 (a circumstance in which IP-10 antibody was estimated to remain in excess) reduced but did not completely ablate IP-10 staining (data not shown). The reduction in IP-10 staining was even more pronounced when the IP-10 antibody was preadsorbed with 500 ng/ml rhIP-10 (a circumstance in which the blocking protein was estimated to be in molar excess; Fig. 5A and 5B ).



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  		Figure 5. Immuno-alkaline phosphatase/FastRed staining of IP-10 in HCV-infected and uninfected liver can be blocked using rhIP-10 protein. (A) In the chronic HCV-infected liver, IP-10 is strongly expressed by hepatocytes, particularly around areas of mononuclear cell aggregates (original magnification, x400). (B) Preadsorbing the IP-10 antibody with 500 ng/ml rh-IP-10 protein blocked the majority of cytoplasm-associated hepatocyte staining. Note, however, the presence of nonspecific staining along sinusoidal margins (original magnification, x400). (C) Uninfected liver stained with 5 µg/ml IP-10 reveals little or no staining by hepatocytes. (D) Uninfected liver stained with 5 µg/ml IP-10 preadsorbed with 100 ng/ml rh-IP-10 protein demonstrates nonspecific staining along sinusoidal margins. (E) Chronic HCV liver and (F) uninfected liver sections were stained with polyclonal goat IgG (5 µg/ml) and were used as negative controls (original magnification, x200). Shown are representative data from three experiments repeated at least twice. Results were consistent and reproducible.

 
It is interesting that the reduction in specific anti-IP-10 staining following preadsorption with rhIP-10 was paralleled by an increase in nonspecific staining along sinusoidal margins. This artifact was observed in HCV-infected liver (Fig. 5A and 5B) , in which strong, intrinsic anti-IP-10 staining is evident, and noninfected explant liver, in which anti-IP-10 staining is absent or weak (Fig. 5C and 5D) . IP-10 antibody specificity was further confirmed in a second tissue type (inflamed tonsil). Tonsil sections stained for IP-10 showed large numbers of positively staining mononuclear cells in parafollicular (T cell) areas, particularly around germinal centers. Preadsorption of the anti-IP-10 antibody with rh-IP-10 reduced the intensity and number of positive-staining cells in a concentration-dependent manner, thereby confirming that the staining was specific (Fig. 6A 6B 6C 6D ).



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  		Figure 6. Immunohistochemical staining of IP-10 in the tonsil can be blocked using rhIP-10 protein. Sections were stained as described in Materials and Methods. (A) In the tonsil, IP-10 is strongly expressed by mononuclear cells in the parafollicular and medullary regions. (B) IP-10 expression was partially blocked by preabsorbing the anti-human IP-10 antibody with 100 ng/ml rhIP-10 (antibody in excess; original magnification, x400) and (C) completely blocked by preadsorbing with 500 ng/ml rhIP-10 (protein in excess; original magnification, x400). (D) Tonsil stained with polyclonal goat IgG (5 µg/ml) was used as the negative control (original magnification, x200). Shown are representative data from three different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report confirms that IP-10 is up-regulated in the liver during chronic HCV infection and is the first study to demonstrate a correlation between IP-10 expression and the accumulation of CXCR3 expressing T lymphocytes in the liver. Further, it is the first study illustrating IP-10 expression by hepatocytes in humans as determined by immunohistochemistry.

The data in this study are concordant with recent publications demonstrating an increase in the level of IP-10 expression in the liver during chronic HCV infection and extend these findings by quantifying the level of IP-10 mRNA in the liver and correlating these findings with histological parameters [17 , 19 ]. IP-10 mRNA levels are a likely correlate of IP-10 protein expression, as demonstrated in a previous study by Boorsma et al. [23 ]. This study provides the novel and important finding that IP-10 mRNA levels strongly correlate with the histological grading of the liver injury and more specifically, with the lobular necroinflammatory score. Phenotypic analysis of the lobular infiltrate demonstrated that T lymphocytes were the predominant cell type in this location. Work conducted in our own laboratory and by others has confirmed that these cells are almost exclusively CD8+ [11 , 24 , 25 ]. By contrast, no correlation was found between IP-10 expression and the portal inflammation component of the Scheuer score. This finding is consistent with the fact that the portal zone contains a mixed infiltrate of lymphocytes, not all of which are responsive to IP-10 (e.g., B lymphocytes). No correlation was found between the numbers of B lymphocytes or CD68+ macrophages and elements of the Scheuer score, suggesting they do not play an important role in ongoing liver cell injury. The factors regulating B cell recruitment to the liver are currently unknown. These findings provide further evidence for the role of IP-10 in the pathogenesis of HCV infection, as lobular activity is associated with progressive liver disease and a poorer prognosis [26 27 28 29 ].

IP-10 recruits T lymphocyte subsets expressing the CXCR3 receptor, including activated T lymphocytes of the T helper type 1 phenotype, a proportion of which are likely to be antigen-specific CD4+ cells [30 ]. In addition, the likely effector cells of antiviral immunity, CD8+ cytotoxic T cells and NK cells, also express CXCR3 and are responsive to IP-10 in chemotaxis [31 32 33 ].

In this study, T lymphocytes in the blood were significantly less likely to express CXCR3 compared with T lymphocytes in the liver, where they were almost uniformly CXCR3-positive. This was observed for CD4 and CD8 T lymphocytes. Further, T lymphocytes expressing CXCR3 in the liver could be distinguished from their counterparts in the blood on the basis of CD69 expression, a marker of recently activated cells. This finding is consistent with reports that naïve lymphocytes are rarely found in nonlymphoid tissues [34 , 35 ]. Together, these data support the notion that the CXCR3/IP-10 pathway is important for the recruitment of T lymphocytes in the liver during HCV infection. Alternatively, the enrichment of CXCR3+ T lymphocytes in the liver might be a result of induced CXCR3 expression after T cells enter the liver using a non-IP-10-dependent mechanism. Although this seems unlikely, the significant expression of IP-10 in the infected liver in this case may provide important retention signals, resulting in the observed accumulation of this T cell phenotype. Other signals likely to enhance the retention and survival of T lymphocytes in the liver include the significant expression of other CXCR3 ligands, particularly monokine induced by IFN-{gamma}. In addition, further signals are likely to be provided by cellular interactions with B lymphocytes and antigen presenting cells, which have been observed to form organized follicle-like bodies in portal tracts and the proinflammatory cytokine milieu of the virally infected liver.

This study also sought to examine the expression of IP-10 protein and define by immunohistochemistry the cellular source of IP-10 in the HCV-infected liver. Recent studies have variously indicated hepatocytes or sinusoidal endothelial cells as the main source of IP-10 in the HCV-infected liver [17 , 19 ]. In this investigation, examination of liver biopsy sections revealed varying levels of IP-10 expressed by hepatocytes in a diffuse pattern throughout the liver lobule. The intensity of IP-10 staining, similar to IP-10 mRNA expression, appeared to correlate with the histological and inflammatory grading of the biopsy. The most intense staining was seen in hepatocytes adjacent to mononuclear cell aggregates. No other cell types were found to express IP-10 with the exception of a small number of mononuclear cells observed in an expanded portal tract from one patient with severe portal and lobular inflammation. These results are consistent with previous data from in situ hybridization studies examining IP-10 mRNA expression in patients with chronic HCV infection and murine models of hepatitis [19 , 36 ]. In addition, rat hepatocytes have been demonstrated to express IP-10 by immunohistochemistry following ischaemia/reperfusion of the liver [37 ].

However, our findings are at variance with those of Shields et al. [17 ], who observed immunoreactivity for IP-10 exclusively in sinusoidal endothelial cells. The same polyclonal anti-human IP-10 antibody was used in the experiments presented here, and the concentration of this primary antibody was comparable in instances. Hence, these conflicting results are not easily explained by variations in the specificity of different primary anti-IP-10 antibodies or antigen/antibody stoichiometry. The blocking experiments reported here, with preadsorbtion of the primary antibody with rhIP-10 protein, confirm that the primary antibody was indeed binding IP-10 and that hepatocytes were the cellular source. The specificity of the primary antibody for IP-10 was confirmed in a second tissue type (inflamed tonsil) with an identical outcome. The only apparent staining of sinusoidal endothelial cells evident in the current report was subsequently shown to be artifactual, as it only occurred in the presence of IP-10 antigen/IP-10 antibody complexes and could be reproduced in HCV-uninfected explant liver. This nonspecific staining might be explained by the binding of recombinant IP-10 protein, complexed with the detection antibody, to IP-10 binding sites in the tissue. Endothelial cells have at least two such binding sites: Cell-surface heparan sulfate proteoglycan is abundantly expressed on endothelial cells and contains a common binding site for IP-10 and platelet factor-4 [38 ]. Furthermore, recent data demonstrate that the IP-10 receptor, CXCR3, is expressed by endothelial cells in inflamed and neoplastic tissues, including endothelial cells of small vessels in the liver of patients with active cirrhosis [39 ]. However, it is unclear to what extent such receptors might be "available" for binding of the immune complex (IP-10/anti-IP-10 antibody) in formalin-fixed paraffin-embedded tissue after antigen retrieval. Taken together, these findings strongly implicate hepatocytes, rather than sinusoidal endothelial cells, as the primary source of IP-10 in the liver.

This result is particularly significant, as it raises the question as to whether HCV infection of hepatocytes directly induces IP-10 expression. Although this remains to be demonstrated, evidence from other viral infections suggests it is possible. A number of viruses and viral gene products, including measles virus and the human immunodeficiency virus-1 tat protein, have been shown to induce IP-10 expression in vitro, particularly by cells of the central nervous system such as astrocytes and glial cells [40 , 41 ]. The induction of IP-10 expression by HCV-infected hepatocytes provides one possible mechanism to explain the significant level of IP-10 expression observed in HCV-infected liver biopsy samples. It is interesting that the percentage of hepatocytes observed in this study to be expressing IP-10 is comparable with the percentage of hepatocytes demonstrated to be infected with HCV in recent reports (mean, 46%; range, 15–81%) [42 ]. Future studies examining coexpression of HCV proteins and IP-10 in hepatocytes of patients with chronic HCV infection are planned. Alternatively, such expression may be triggered by IFN-{gamma}, produced by infiltrating T lymphocytes or NK cells.

In summary, IP-10 is abundantly expressed in the liver of patients with chronic HCV and is produced by hepatocytes. The level of IP-10 mRNA expression correlated with the severity of the T cell predominant lobular inflammation, suggesting that this chemokine regulates T lymphocyte recruitment. Liver-infiltrating T lymphocytes uniformly expressed the IP-10 receptor. As lobular inflammation is an increasingly recognized correlate of disease progression in chronic HCV infection, this suggests that IP-10 may be an important component of antiviral effector mechanisms and hepatocellular injury via recruitment of CD8+ cytotoxic T cells to the vicinity of HCV-infected hepatocytes. The development of reagents to manipulate the IP-10/CXCR3 trafficking pathway may be a useful, therapeutic strategy for hepatitis C infection.


   ACKNOWLEDGEMENTS
 
C. E. H. and J. J. P. were supported in this work by postgraduate scholarships from the National Health and Medical Research Council (NHMRC) of Australia. We thank Dr. P. Bullpitt for his histological assessment of liver biopsy tissue. We also thank Dr. W. Rawlinson and Dr. P. White for viral load data. The Viral Hepatitis Research Unit, the Inflammation Research Unit, and Department of Immunology and Infectious Diseases are all members of the Australian Centre for Viral Hepatitis.

Received March 7, 2003; revised May 4, 2003; accepted May 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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