Expression of Cytokines (TNF-, IL-1
, and IL-2) in Chronic Hepatitis C
:
Comparative Hybridocytochemical and Immunocytochemical Study in Children and Adult Patients
awa Biczysko
Spachacz
Department of Histology and Embryology, University of Medical Sciences, Pozna, Poland (AK,MZ,RS,JSZ); Department of Histology, University of Medical Sciences, Wroc
aw, Poland (MZ); Department of Clinical Pathomorphology, University of Medical Sciences, Pozna
, Poland (WB); Department of Health Prophylaxis, University of Medical Sciences, Pozna
, Poland (JW); and Ward of Infectious Diseases, J. Stru
Hospital, Pozna
, Poland (AA)
Correspondence to: Aldona Kasprzak, Dept. of Histology and Embryology, University of Medical Sciences, wi
cickiego 6, 60-781 Pozna
, Poland. E-mail: akasprza{at}amp.edu.pl
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Summary |
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Key Words: chronic hepatitis C TNF- IL-1
IL-2 children adult patients grading staging immunocytochemistry in situ hybridization ImmunoMax technique
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Introduction |
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Because of the lack of comparative studies on cytokine expression in children and in adults with chronic hepatitis C and relatively few literature data on cell sources of the proteins, we have decided to continue our earlier studies (Kasprzak et al. 2002) on the model of chronic hepatitis C infection.
The aim of the study was to investigate the intrahepatic localization of mRNAs for pro-inflammatory cytokines produced mainly by macrophages, TNF- and IL-1
and their respective proteins and the T-helper type 1 (Th1)-associated cytokine IL-2 in liver specimens from children and adults chronically infected with HCV by use of a non-isotopic variety of ISH, ICC alone, and both combined with ImmunoMax technique to determine the cellular source of these cytokines. We planned to compare expression of the mentioned cytokines between children and adults with chronic hepatitis C on the one hand and control individuals on the other. Attempts were also made to correlate the cytokine expression with (a) inflammation activity (grading) and/or advancement of fibrosis (staging), and (b) serum alanine transaminase (ALT) levels in groups of children and adult patients.
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Materials and Methods |
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Tissue Sample Processing
In total, 27 liver biopsy specimens were obtained during routine diagnostic liver biopsies after receiving informed consent from the patients. Tissue specimens were fixed in 10% formalin and embedded in paraffin for light microscopy. Histopathological lesions were evaluated after the classical H+E staining as well as the silver technique and the trichromate technique according to Masson and periodic acidSchiff with diastase pretreatment. At least 10 sections were prepared from each biopsy. Each biopsy specimen was evaluated based on a simple numerical scoring system for the grade of portal/periportal necroinflammation (G1) (04), the grade of lobular necroinflammation (G2) (04), and for the stage of fibrosis (S) (04), as proposed by Scheuer et al. (2002). The histological lesions considered to be characteristic of chronic hepatitis were also evaluated. Histological evaluation was performed independently by two experienced histopathologists.
In Situ Hybridization
For the studies, 5-µm-thick sections were cut and mounted on SuperFrost/Plus microscope slides from the total of 27 liver biopsies. Two digoxygenin-labeled oligoprobes specific for human cytokine mRNA were used, including (a) human TNF- probe cocktail (R&D Systems; Minneapolis, MN), (b) human IL-1
probe cocktail (R&D Systems), detected with sheep anti-digoxygenin monoclonal antibodies (MAbs) (Fab fragments), labeled with horseradish peroxidase (Roche). In the hybridocytochemical studies, the classical ones and those amplified by the ImmunoMax technique (Komminoth and Werner 1997
), sequential sections of the tissue material were applied, which was pre-tested by ICC techniques. The ISH protocol of R&D Systems was employed with our own modification and ImmunoMax amplification of the signal. Subsequent stages involved paraffin removal from the section using xylene, ethanols, and washing in diethyl pyrocarbonate (DEPC) (Sigma Chemical; St Louis, MO)-treated distilled water, each lasting 15 min, and washing in 0.05 M Tris (AccuGene; Landover, MD) for 5 min at room temperature (RT). Endogenous peroxidase was blocked by a 15-min incubation of the section in 1% H2O2 at RT. Then the sections were digested with proteinase K (DAKO; Carpinteria, CA) 100200 µg/ml for 1015 min at 37C. Next, the sections were washed in 1 x PBS for 10 min at RT and fixed again in 0.4% PFA at 4C for 15 min. They were washed in DEPCdistilled water for 1 min each. The prehybridization solution (Sigma) was applied under a coverslip and the preparation was incubated for around 1 hr at 37C. Various concentrations of the oligoprobe were tested, ranging from 50 to 500 ng/ml, dissolved in the prehybridization solution plus 30% formamide. The hybridization itself took place overnight (1618 hr) at around 38C under a coverslip in a humid chamber. After removal of the coverslips, the sections were washed in decreasing concentrations of SSC (Promega; Madison, WI) plus 30% formamide (Sigma), and then in 0.1 M Tris-HCl, pH 7.6, and 0.1% Triton X-100 in TBS for 15 min at RT. Sheep anti-digoxygenin MAbhorse radish peroxidase (HRP) (Roche) complex was applied for around 45 min at RT at a dilution of 1:30. After a wash in PBS/Tween-20, biotinylated tyramine (PerkinElmer Life Sciences; Boston, MA) was applied at 1:50 dilution for around 3 min at RT. The streptavidinperoxidase complex was added for 30 min at RT. The hybrids were detected using peroxidase substrate, 0.05% DAB (DAKO) in 0.05 M Tris-HCl, pH 7.6, supplemented with 0.001% H2O2 for 5 min at RT. Some preparations were counterstained with hematoxylin and mounted with a glycerol-based mounting medium. As controls for ISH, lymph nodes from three healthy donors served as initial positive controls. Negative controls included (a) hybridization without addition of the molecular probe and (b) incubations of slides in an RNase A solution (R&D Systems), 20 mg/ml in 2 x SSC/10 mM MgCl2, pH 8, for 30 min at RT before hybridization. All the experiments were performed at least twice.
Immunohistochemistry
The following specific MAbs were used: (a) mouse anti-human TNF- antibody (R&D Systems), (b) goat anti-human IL-1
antibody (R&D Systems), (c) mouse anti-IL-2 antibody (R&D Systems), (d) mouse anti-human macrophage antibody CD68 (DAKO), and (e) anti-human Von Willebrand Factor antibody (DAKO Epos; Glostrup, Denmark). The first stages of the ICC reaction, up to blocking of endogenous peroxidase, followed the procedure applied for hybridocytochemistry. Then the sections were treated with primary MAb at 1:20 dilution (anti-IL-1
) and 1:100 dilution (anti-TNF-
and anti-IL-2) or ready-for-use dilutions (anti-CD68 and anti-human Von Willebrand Factor) overnight at 4C, then with the secondary biotinylated link of anti-mouse and anti-rabbit IgG and with the streptavidinbiotinperoxidase complex (ABC) (DAKO). The studies followed the classical ABC technique (Hsu et al. 1981
), alone or associated with the ImmunoMax technique (Komminoth and Werner 1997
). In our studies we did not use antigen retrieval but amplification of the final signal using biotinylated tyramine. In the technique, termed the ImmunoMax technique, the key reaction involved a 3-min incubation with biotinylated tyramine (PerkinElmer) at 1:50 dilution at RT. This was followed by another application of the streptavidin complex. The color reaction was evoked with the HRP substrate 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO) in 0.05 M Tris-HCl buffer, pH 7.6, supplemented with 0.001% H2O2. In tissue sections, endogenous peroxidase was blocked with 1% H2O2. Control reactions employed control sera of the respective species in 0.05 M Tris-HCl, pH 7.6, supplemented with 0.1% BSA and 15 mM sodium azide (negative control) (DAKO). Most of the histological preparations were subjected to double ICC reactions, including a macrophage marker (CD68) or a marker for endothelial cells (Von Willebrand Factor) and one of the cytokines. The terminal reaction product was visualized with DAB (brownish-black signal) or DAB supplemented with nickel (blue-gray colour). Some preparations were counterstained with hematoxylin and mounted with a glycerol-based mounting medium.
Quantification of ISH and ICC Results and Statistical Analysis
The contents of cytokines and of respective mRNAs in liver biopsies demonstrated using hybridocytochemistry and the ABC method, both combined with the ImmunoMax technique, were evaluated by the semiquantitative technique, relating the score of 0 to 4 points to the fraction of stained cells (score 0, 0% cells; 1, less than 5% cells; 2, 520% cells; 3, 2040%; and 4, more than 40% positive cells). It should be mentioned that semiquantitative evaluation was conducted exclusively in preparations obtained using the ImmunoMax technique (both in ICC and in ISH), which yielded an intense color reaction. The preparations were examined under a light microscope at x400 magnification. In each section of the liver biopsy, five fields in hepatic lobules and the periportal area and at least five different portal spaces in each specimen were examined. The calculations were performed on the mean of three consecutive sections of liver biopsy material from HCV-infected patients and resulted in mean numbers separate for the lobular/periportal area and the portal area. Cellular localization of the studied proteins and respective transcripts was evaluated (cell nucleus, cytoplasm, membranes).
StatXact Software (Cytel Software; Cambridge, MA) was used for statistical analysis of small data sets. To determine statistical significance of variations in percentage of cells with transcripts and proteins studied using the ImmunoMax technique, we first calculated the mean values of immunostaining or hybridocytochemical signal scores for both liver biopsy groups (adults and children) as well as control biopsy specimens, separately for the lobular/periportal area and the portal spaces. The values were then compared using the MannWhitney U-test for nonparametric independent data and the Wilcoxon test for nonparametric dependent data. Correlations between data rows were determined with Spearman's rank correlation index. In determining the correlations, means of the total expression of cytokine mRNA or of the respective protein were taken into account (i.e., expression in the lobules, periportal and portal spaces) and related to the sum of grading (G1+G2), staging (S), and ALT, respectively, in the groups of children and adult patients.
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Results |
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Discussion |
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For the present studies, three cytokines were selected (TNF-, IL-1
, and IL-2). TNF-
is required for normal proliferation of hepatocytes in liver regeneration and it exhibits anti-apoptotic activity (Simpson et al. 1997
) but, on the other hand, it represents a well-recognized mediator of hepatocyte death (Zhu et al. 1998
). IL-1 plays other roles in the liver, including induction of the acute-phase response and autoactivation of Kupffer cells (Winwood and Arthur 1993
). IL-2 is produced mainly by Th1 cells and its role involves stimulation of proliferation and activation of most T-lymphocytes, NK-cells, and B-lymphocytes. IL-2 is also known to function in some non-lymphoid cells (reviewed in Smith 1984
; Peters 1996
). Moreover, normal and neoplastically transformed epithelial cells can express endogenous IL-2, which is processed and secreted in the same way as in hematopoietic cells (Reichert et al. 1998
). The few authors who have examined intracellular expression of cytokines in chronic hepatitis C in vivo mention liver sinusoidal cells and cells of inflammatory infiltrates as the most common sources of the cytokines (GonzalezAmaro et al. 1994
; Dumoulin et al. 1999
; Oyanagi et al. 1999
). Under certain conditions, other cells, such as endothelial cells, HSCs, bile duct epithelial cells, and hepatocytes are able to synthesize TNF-
and IL-1 (GonzalezAmaro et al. 1994
; Tsukamoto 1999
; Kasprzak et al. 2002
). Opinions on the amount of cytokines detected in livers with chronic hepatitis C or on correlation of cytokine expression on the one hand and grading and/or staging and/or intrahepatic viral load on the other are divergent (GonzalezAmaro et al. 1994
; Larrea et al. 1996
; Napoli et al. 1996
; Dumoulin et al. 1999
; Kinnman et al. 2000
). No comparative studies have been conducted on intrahepatic expression of cytokines in chronic hepatitis C in children or adults, particularly those employing direct visualization of transcripts and/or proteins at the cellular level. Compared to control biopsies, our studies have demonstrated more pronounced expression of IL-1
mRNA in both groups of HCV-infected patients and TNF-
mRNA in adult patients, as well as of all cytokines in children and in adults with chronic hepatitis C. Comparison of cytokine expression in children with that in adults has demonstrated more intralobular IL-1
in adults, but total expression (in lobules and in portal spaces) did not differ between the two groups of patients, similarly to expression of TNF-
and IL-2. Augmented expression of transcripts for the two cytokines has been observed mainly in cells of liver sinusoids (macrophages and endothelial cells), in occasional cells of inflammatory infiltrate, and in a few hepatocytes, which has been partially consistent with earlier reports (Gonzalez Amaro et al. 1994
; Larrea et al. 1996
; McGuinness et al. 2000
). A similar localization of transcripts for cytokines in chronic HCV infection has also been noted by Dumoulin et al. (1999)
, but in their hands IL-1ß immunoreactivity and mRNA levels were less pronounced in chronic HCV than in control samples. In contrast, no significant differences in TNF-
mRNA levels were observed between chronic hepatitis C samples, primary biliary cirrhosis (PBC), or control samples (Dumoulin et al. 1999
). Compared to our observations, the differences might be explained by the fact that the authors studied cytokine expression in a relatively large group of patients with hemophilia. Even though we have observed a higher percentage of cytokine-producing cells in the liver sinusoids compared to controls, the observation has been paralleled by detection of a markedly lower percentage of cells with TNF-
and IL-1
transcripts compared to the amounts of the respective cytokine proteins, and unsuspected localization of the latter has involved mainly hepatocytes rather than cells in liver sinusoids. The participation of hepatocytes in production of cytokines in viral hepatitis has been documented with increasing frequency (Gonzalez Amaro et al. 1994
; Oyanagi et al. 1999
; Kasprzak et al. 2002
). TNF-
biosynthesis is regulated at transcriptional and post-transcriptional levels, and it is possible that TNF-
is processed by a different pathway in hepatocytes than in macrophages or T-lymphocytes (Kriegler et al. 1988
; Beutler and Cerami 1989
; Santis et al. 1992
). Our studies have demonstrated expression of additional cytokines, i.e., IL-1
and IL-2, in HCV-infected hepatocytes. A probable although still unclear hypothesis remains their participation in the pathogenesis of infection. According to some authors, in patients with chronic hepatitis C the elevated serum TNF-
reflects the intensity of inflammatory lesions (grading) and decreases after anti-viral treatment (Neuman et al. 2002
). In both groups of our patients, no correlation was observed between grading and cytokine expression, despite higher grading in adults. This points to participation of additional cytokines or other factors in intensification of inflammation. A significant involvement of TNF-
has also been suggested in the process of liver fibrosis in chronic hepatitis C (Kinnman et al. 2000
). Both TNF-
, and IL-1 are known to belong to the group of cytokines capable of stimulating proliferation of Ito cells (HSCs), cells of key importance in the process of liver fibrosis (Tsukamoto 1999
). Only in our adult patients with chronic hepatitis C has positive correlation been observed between TNF-
and staging. However, participation of other pathogenic factors (in addition to HCV and elevated TNF-
) in production of higher staging in adults cannot be excluded. Our studies have demonstrated a particularly high expression of IL-2 in hepatic lobules, both in children and in adult patients with chronic hepatitis C, compared to TNF-
and, in the case of children, also to IL-1
. The augmented IL-2 expression in patients with chronic hepatitis C is believed to provide evidence for an intrahepatic response of Th1, but cell sources of the cytokine have not always been documented (Napoli et al. 1996
; Dumoulin et al. 1997
; McCaughan et al. 2000
). In our study, expression of the IL-2 dominated in hepatocytes, which has been unsuspected and, until now, such a high expression of the cytokine in liver lobules (and in hepatocytes) has found no confirmation in the literature. The role of augmented IL-2 expression in hepatocytes of patients with chronic hepatitis C requires further studies, including those using techniques of molecular biology. The very low expression of cytokines in cells of inflammatory infiltrates in our patients (both in portal spaces and in the lobules) might reflect weak stimulation of mononuclear cells, low numbers of infiltrating activated macrophages (CD68), or disturbed production of pro-inflammatory cytokines by cells of the infiltrate. Studies performed in vitro using the protein NS3 to stimulate responses of T-cells from infected individuals have shown both an absence of IL-2 protein secretion by the lymphocytes and a failure to amplify IL-2 mRNA on stimulation (Eckels et al. 1999
). Other authors have documented a correlation between increased IL-2 expression and increased IFN-
expression or grading/staging in chronic hepatitis C infection (Napoli et al. 1996
). Our studies have not confirmed this observation.
For studies on cytokine localization in liver, this study has for the first time employed the ImmunoMax technique, which has increased sensitivity above the classical ICC and ISH tests. Compared to controls, augmented expression of TNF- , IL-1
, and IL-2 has been demonstrated in patients with chronic hepatitis C. On the one hand this indicates the role of cytokines in the chronic inflammatory process and, probably, also in a mechanism of liver regeneration. On the other hand, it reflects inefficient anti-viral defense. The comparable level of expression of the cytokines in HCV-infected children and adults, confronted with higher grading and staging noted in adult patients, points to participation of additional cytokines or other factors that intensify degeneration of the organ in HCV-infected adults. On the basis of our results, it is difficult to clarify causes of the high expression of proteins of the three cytokines in hepatocytes and only low levels of transcripts of such a localization. The result suggests that, in addition to the classical ones, other pathways of cytokine expression operate in various cells, e.g., under the effects of active HCV replication.
In this study, cellular localization has for the first time been compared and expression of TNF-, IL-1
, and IL-2 has been semiquantitatively appraised in children and adults with chronic hepatitis C. We have confirmed expression of TNF-
in hepatocytes and, in addition, we have demonstrated the potential for IL-1
and IL-2 expression in the cells. Chronic hepatitis C is accompanied by augmented expression of cytokines such as TNF-
, IL-1
, and IL-2, comparable in groups of children and of adult patients. Nevertheless, the augmented expression remains insufficient to terminate the infection and this may be linked to the comparatively high frequency of chronic transformation of HCV infection in the two groups of patients.
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Acknowledgments |
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Footnotes |
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