Cancer Research Annual Meeting 2010 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kaneko, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kaneko, S.
[Cancer Research 64, 3326-3333, May 1, 2004]
© 2004 American Association for Cancer Research

Immunology

Different Procarcinogenic Potentials of Lymphocyte Subsets in a Transgenic Mouse Model of Chronic Hepatitis B

Yasunari Nakamoto1, Takashi Suda2, Takashi Momoi3 and Shuichi Kaneko1

1 Department of Gastroenterology, Graduate School of Medicine, 2 Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Kanazawa, and 3 Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immune response to hepatitis viruses is believed to be involved in the development of chronic hepatitis; however, its pathogenetic potential has not been clearly defined. The current study, using a transgenic mouse model of chronic hepatitis B, was designed to determine the relative contributions of the immune cell subsets to the progression of liver disease that induces hepatocellular carcinogenesis. Hepatitis B virus transgenic mice were adoptively transferred with CD4+ and CD8+ T cell-enriched or -depleted and B cell-depleted splenocytes obtained from hepatitis B surface antigen-primed, syngeneic nontransgenic donors. The resultant liver disease, hepatocyte apoptosis, regeneration, and tumor development were assessed and compared with the manifestations in mice that had received unfractionated spleen cells. Transfer of CD8+-enriched splenocytes caused prolonged disease kinetics, and a marked increase in the extent of hepatocyte apoptosis and regeneration. In 12 of 14 mice the transfer resulted in multiple hepatocellular carcinomas (HCCs) comparable with the manifestations seen in the mice transferred with total splenocytes. In contrast, mice that had received CD4+-enriched cells demonstrated lower levels of liver disease and developed fewer incidences of HCC (4 of 17). The experiment also revealed that all of the groups of mice complicated with HCC developed comparable mean numbers and sizes of tumors. B-cell depletion had no effect on disease kinetics in this model. Taken together, these results demonstrate that the pathogenetic events induced by CD8+ T-cell subset are primarily responsible for the induction of chronic liver disease that increases tumor incidence, suggesting their potential in triggering the process of hepatocarcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular carcinoma (HCC) occurs after many years of chronic hepatitis (1 , 2) . During the process, both viral and host factors have been implicated in liver cell transformation and carcinogenesis. On the one hand, some viral proteins, i.e., hepatitis B virus (HBV) X protein (3 , 4) and hepatitis C virus core protein (5 , 6) , are considered to contribute to tumor development in the liver, because high-level expression of the proteins increases the incidence of HCC in transgenic mice. Furthermore, most tumors contain clonally integrated HBV DNA and microdeletions in the flanking cellular DNA, which could theoretically deregulate cellular growth control mechanisms (7) . And COOH-terminally truncated viral envelop proteins expressed from integrated subviral DNA may have transactivating activity (8 , 9) and could potentially contribute to carcinogenesis in chronic HBV infection.

On the other hand, prolonged inflammation is thought to set up a cycle of liver cell destruction and regeneration, resulting in a mitogenic and mutagenic environment that can precipitate random genetic and chromosomal damage, and lead to the development of HCC (10, 11, 12) . In patients with chronic hepatitis B and C, CD4+ and CD8+ T lymphocytes specific for the viruses are detectable in the peripheral blood and in intrahepatic infiltrates, and are suggested to play a role in the immune pathogenesis of liver disease (13, 14, 15, 16) . Furthermore, transfer of the virus-specific CD4+ and CD8+ T-cell clones was observed to induce acute necroinflammatory liver disease in the models of HBV transgenic mice (17, 18, 19, 20) . However, the relative contribution of CD4+ and CD8+ T lymphocytes to the induction of chronic liver cell injury was not determined, because the T-cell clones induced neither prolonged liver diseases nor HCC in the models of acute hepatocellular injury, and because the animal model that pathophysiologically reproduces human chronic viral hepatitis was not available. In an effort to clarify the carcinogenic potential of chronic inflammation, we have developed a model of chronic immune-mediated liver disease using HBV transgenic mice that express the envelop proteins in the hepatocytes (21) . The results demonstrate that continuous intrahepatic inflammation is sufficient to cause liver cancer in the absence of pre-existing viral transactivation, insertional mutagenesis, or genotoxic chemicals during chronic HBV infection.

We have shown recently that the administration of anti-Fas ligand (FasL) neutralizing antibody reduces hepatocyte apoptosis, proliferation, and liver inflammation, and eventually diminishes the development of HCC. This observation suggests a critical involvement of FasL-induced pathogenetic events in the process of hepatocarcinogenesis (22) . We have also reported evidence that the FasL-dependent pathway is critically involved in the development of acute liver cell injury induced by CD8+ cytotoxic T-lymphocyte (CTL) clones (23 , 24) . Because FasL is known to be expressed on activated T lymphocytes (25, 26, 27, 28, 29) , we speculated that the CD8+ T-cell subset was implicated as a causative factor responsible for the chronic liver cell injury that promotes hepatocarcinogenesis.

To determine the involvement of immune cell subsets in the progression of chronic liver disease, the current experiment was performed in the model of chronic immune-mediated hepatitis in which T- and B-cell subset-depleted or -enriched splenocytes obtained from HBV-primed, nontransgenic mice were transferred into the transgenic recipients, and liver disease and tumor development were monitored. The results demonstrate that each cell subset causes unique kinetics of liver disease and different incidence of liver cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
HBV Transgenic Mice.
Hepatitis B surface antigen (HBsAg) transgenic mouse lineage 107–5D [official designation Tg(Alb-1, HBV)Bri66; inbred B10D2, H-2d ] was kindly provided by Dr. Francis V. Chisari (The Scripps Research Institute, La Jolla, CA; Ref. 30 ). Lineage 107–5D contains the entire HBV envelope coding region (subtype ayw) under the constitutive transcriptional control of the mouse albumin promoter (30) . These mice express the HBV small, middle, and large envelope proteins in their hepatocytes (30 , 31) , they are immunologically tolerant to HBsAg at the T-cell level (32) , and they display no evidence of liver disease during their lifetime although they do develop "ground glass" hepatocytes due to overexpression of the large envelope protein (30) . There is no X-RNA or X-protein expression detectable in the livers of these animals.4 Importantly, the mice develop a severe MHC class I-restricted necroinflammatory liver disease after the adoptive transfer of HBsAg-specific CTLs (17 , 18 , 30) .

Disease Model.
To break tolerance at the B- and T-cell levels, HBV transgenic mice were thymectomized, bone marrow-reconstituted, and adoptively transferred with nontransgenic immune systems according to the procedures described previously (21 , 33) . Briefly, 8–10-week-old male transgenic mice were thymectomized. Seven days later the mice were irradiated (900 cGy) and then reconstituted by the i.v. injection of 107 bone marrow cells collected from the femurs and tibias of syngeneic nontransgenic B10D2 (H-2d) mice. One week after the bone marrow transfer, the animals were injected i.v. with the indicated numbers of splenocyte subsets from nontransgenic B10D2 (H-2d) mice that were infected i.p. with a recombinant vaccinia virus expressing HBsAg (HBs-vac) 3 weeks before the splenocyte transfer (17) . At the same time, transfer of total splenocytes from the primed nontransgenic mice and the unprimed transgenic littermates were performed for control purposes. The resultant hepatocellular injury was monitored biochemically as serum alanine aminotransferase (ALT) activity (10) . Results were expressed as mean units per liter ± SE of serum ALT activity, and differences between groups were assessed for statistical significance by Student’s t test. Tumor development was assessed by abdominal palpation and confirmed by autopsy at which time the number of tumors visible at the surface of each liver was counted, and the diameter of each tumor was measured with a millimeter rule. All of the experiments satisfied the Guidelines for the Care and Use of Laboratory Animals in Takara-machi Campus of Kanazawa University.

Depletion and Enrichment of T- and B-Cell Subsets.
To deplete CD4+ or CD8+ T cells, splenocytes were treated with monoclonal antibodies (mAbs) specific for CD4 (GK1.5) or CD8 (2.43; American Type Culture Collection, Manassas, VA), respectively, and then with rabbit complement (Cedarlane, Hornby, Ontario, Canada). B cells of splenocytes were depleted on the Mouse T Cell immunocolumn (Cytovax, Edmonton, Alberta, Canada), by treatment with mAb specific for MHC class II I-Ad (MK-D6; American Type Culture Collection), and with rabbit complement. To enrich CD4+ or CD8+ T cells, splenocytes were passed over the Mouse T Cell immunocolumn and treated with a combination of anti-CD8 and anti-I-Ad mAbs, or anti-CD4 and anti-I-Ad mAbs, respectively, and then with rabbit complement. The purity of the T- and B-cell populations was monitored by immunolabeling with FITC-conjugated rat mAb specific for mouse CD4 (RM4–5; BD PharMingen, San Diego, CA), and phycoerythrin-conjugated rat mAb specific for mouse CD8 (53–6.7), or CD19, which is a B cell-specific transmembrane protein (1D3; BD PharMingen), followed by fluorescence-activated cell sorter analysis.

Immunohistochemical Analysis.
Tissue samples were fixed in buffered zinc formalin (Anatech Ltd., Battle Creek, MI), embedded in paraffin, sectioned (at 3 µ m), and stained with H&E as described previously (21) . Some of the paraffin sections were treated with anti-proliferating cell nuclear antigen (PCNA) and anti-HBsAg primary solutions (Dako, Carpinteria, CA) at 1:10 and 1:1000 dilutions, respectively, followed by biotin-conjugated secondary antibody (Vector Laboratories, Inc., Burlingame, CA; Ref. 34 ). PCNA+ and HBsAg+ cells were then visualized using a VECTASTAIN ABC Standard kit (Vector Laboratories), and the tissue sections were counterstained with hematoxylin before mounting. Liver tissue samples were also embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen in liquid nitrogen. Cryostat sections of frozen tissues were fixed in 4% paraformaldehyde overnight at 4°C. After blocking biotin, the tissue sections were incubated with rabbit antimouse active caspase-3 antibodies (35) at a 1:400 dilution for 30 min at room temperature, followed by biotin-conjugated goat antirabbit IgG secondary antibodies (Vector Laboratories). The reaction was visualized in the same way as the PCNA staining described above. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) analysis was performed on serial liver sections according to the manufacturer’s instructions (Roche, Indianapolis, IN).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cellular Basis for Prolonged Chronic Immune-Mediated Hepatitis in HBV Transgenic Mice.
To determine the relative contribution of immune cell subsets to prolonged chronic immune-mediated hepatitis in HBV transgenic mice, the splenocytes isolated from HBsAg-primed nontransgenic mice were depleted (Fig. 1)Citation or enriched (Fig. 2)Citation of CD4+ and CD8+ T cells and CD19+ B cells, and they were then adoptively transferred into thymectomized, bone marrow-reconstituted HBV transgenic recipients. The kinetics of all of the disease manifestations was compared with that caused by total splenocyte transfer. As observed previously (21) , total cell transfer caused prolonged chronic hepatitis (Fig. 1)Citation . Briefly, serum ALT activity increased from preinjection levels of 20–40 units/liter to approximately 2000–4000 units/liter within 7 days after adoptive transfer and fell progressively thereafter. Importantly, the ALT activity never returned to baseline in these animals, remaining at least two to three times above normal throughout the experiment. B cell-depleted splenocytes demonstrated disease kinetics comparable with that seen after total cell transfer. Similarly, CD4+ subset-depleted splenocytes caused acute elevation of serum ALT activity within 7 days after the transfer, and the animals developed persistent liver disease, although the peak of disease activity was lower than that seen after total cell transfer. In contrast, CD8+ subset depletion markedly reduced the peak and diminished the disease activity in the chronic phase later than 7 days. In addition, we assume that a contaminating 1.4% (1.3 x 106) of CD8+ T cells in this CD8+ subset-depleted population may not influence the kinetics of liver disease, because we observed that transfer of 1 x 107 total splenocytes, which contained ~20% (2 x 106) CD8+ T cells, did not cause elevation of ALT in this model.4



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Kinetics of serum alanine aminotransferase (ALT) after transfer of CD4+ T cell-depleted, CD8+ T cell-depleted, or B cell-depleted splenocytes in a transgenic mouse model of chronic hepatitis B. The splenocytes were obtained from hepatitis B surface antigen-primed, syngeneic nontransgenic mice. Nine x 107 cells of the splenocytes depleted of CD4+ [CD4+, 1.4%; CD8+, 20.8%; B (CD19+), 32.4%], CD8+ (CD4+, 15.0%; CD8+, 1.4%; B, 28.5%), or B cells (CD4+, 17.5%; CD8+, 38.5%; B, 0.1%) were transferred into hepatitis B virus transgenic mice. At the same time, transfer of the total splenocytes (1 x 108 cells; CD4+, 12.7%; CD8+, 16.7%; B, 26.8%) was performed for control purposes. The serum ALT activity (mean units/liter) was monitored to evaluate liver injury; bars, ±SE. Each group represents 5 animals.

 


View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Kinetics of serum alanine aminotransferase (ALT) after transfer of CD4+ or CD8+ T cell-enriched splenocytes in a transgenic mouse model of chronic hepatitis B. The splenocytes were obtained from hepatitis B surface antigen-primed, syngeneic nontransgenic mice. Five x 107 cells of the splenocytes enriched for CD4+ (CD4+, 78.7%; CD8+, 1.8%; B, 0.9%) or CD8+ (CD4+, 2.0%; CD8+, 84.2%; B, 0.7%) were transfused into hepatitis B virus transgenic mice. At the same time, transfer of total splenocytes from the primed nontransgenic mice (1 x 108 cells; CD4+, 10.4%; CD8+, 21.3%; B, 23.0%) and the unprimed transgenic littermates (1 x 108 cells) were performed for control purposes. The serum ALT activity (mean units/liter) was monitored to evaluate liver injury; bars, ±SE. Each group represents 5 animals. Similar experiments were performed three times and representative data are shown.

 
Consistent with the differences in the kinetics of liver disease induced by the subset-depleted cells, transfer of CD8+ subset-enriched splenocytes displayed the prolonged kinetics comparable with total cell transfer except for the lower peak of ALT (Fig. 2)Citation . In contrast, CD4+ subset-enriched cells caused a transient elevation of ALT 7 days after adoptive transfer that seemed to improve 14 days after the transfer. In addition, we confirmed that transgenic splenocytes induce no disease in the recipient mice, indicating that all subsets of transgenic splenocytes are perfectly immunologically tolerant to the viral antigens as observed previously (21) . Whereas these subset-enriched splenocytes included unidentified non-T and non-B cells that might influence the kinetics of liver disease, the data collectively demonstrate that CD8+ T lymphocytes contributed not only to the induction of hepatocellular injury but also to the maintenance of disease activity in the HBV transgenic mouse model.

In the analysis of liver histology (Fig. 3, A and B)Citation the mice transferred with total splenocytes and with CD8+ subset-enriched cells demonstrated severe infiltration of inflammatory cells, apoptotic and necrotic hepatocytes, many necroinflammatory foci, and mitotic figures. In contrast, the liver samples of mice transferred with the CD4+ subset-enriched cells showed reduced infiltration of inflammatory cells, and minimal induction of apoptosis and liver cell injury. Consistent with the differences in disease activity revealed by the change in the serum ALT level, the results indicate that the CD8+ subset was critically involved in the histological changes of pathological features associated with the establishment of prolonged chronic hepatitis seen in this model.



View larger version (119K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Histopathological features of liver disease caused by transfer of total, CD4+ T cell-enriched or CD8+ T cell-enriched splenocytes in a transgenic mouse model of chronic hepatitis B. Representative mice described in the legend to Fig. 2Citation were killed 7 days after the transfer and analyzed histologically. A, liver specimens of mice that had received total or CD8+ enriched splenocytes show severe infiltration of inflammatory cells, apoptotic (*) and necrotic hepatocytes, many necroinflammatory foci and mitotic cells (arrows). In contrast, liver samples of mice transferred with the CD4+ subset show reduced infiltration of inflammatory cells. H&E; original magnifications: x200 (top) and x300 (bottom). B, quantitative morphometric analysis of apoptotic ({blacksquare}) and mitotic ({square}) hepatocytes. Fifty high-power (x400) fields representing 2 mm2 of liver tissue were examined. Results are expressed means per 1000 hepatocytes; bars, ±SE. Each group represents 3 animals.

 
Apoptosis and Regeneration of Hepatocytes Induced by Differential Immune Cell Subsets in Prolonged Chronic Liver Disease.
To monitor the hepatocyte destruction and regeneration caused by inflammation with immune cell subsets, the activation of the caspase cascade was determined immunohistochemically using mAb specific for the activated form of caspase-3. Degradation of DNA from apo-ptotic hepatocytes was measured by nuclear staining using the TUNEL method, and hepatocyte proliferation was assessed with mAb specific for PCNA (Fig. 4, A and B)Citation . Consistent with the differences in the serum levels of ALT activity, CD8+ subset-enriched splenocytes induced marked caspase-3 activation and DNA degradation, comparable with the effects seen with total splenocytes, whereas CD4 enrichment diminished the number of active caspase-3+ and TUNEL+ hepatocytes. A pair of serial sections in mirror-image orientation stained with antiactive caspase-3 antibodies and the TUNEL method revealed that strong caspase-3 activation and DNA degradation were occurring in hepatocytes along the edge of the area where massive infiltrating cells were found. These data suggest that massive hepatocyte apoptosis may be caused by extensive infiltration of the transferred CD8+ splenocyte subset into the liver tissues. In addition, the observation at the single cell level indicated that the hepatocytes stained by the antiactive caspase-3 and by the TUNEL method did not seem to perfectly coincide. We speculate that this could be due to the difference in the phase of apoptotic process, because caspase activation and DNA degradation may be detected in the earlier and the later phases by the two independent methods, respectively.



View larger version (85K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Immunohistochemical analysis of hepatocellular apoptosis and regeneration induced by transfer of total, CD4+ T cell-enriched or CD8+ T cell-enriched, splenocytes in a transgenic mouse model of chronic hepatitis B. Representative mice described in the legend to Fig. 2Citation were killed 7 days after the transfer. A, immunohistochemical analysis of active caspase-3 and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) analysis were performed in mirror-image serial sections of the liver tissues. TUNEL+ hepatocyte nuclei are indicated with arrows . Proliferating cell nuclear antigen (PCNA) expression was analyzed in the same tissues. Age-matched, untreated hepatitis B virus transgenic mouse liver was stained for PCNA as a control. Original magnifications, x40 (top three panels of caspase-3 and TUNEL staining; bar represents 200 µ m), and x400 (the other panels: bar represents 20 µm). B, quantitative morphometric analysis of caspase-3+ ({blacksquare}), TUNEL+ () and PCNA+ ({square}) hepatocytes. Fifty high-power (x400) fields representing 2 mm2 of liver tissue were examined. Results are expressed means per 1000 hepatocytes; bars, ±SE. Each group represents 3 animals.

 
We also observed many PCNA+ hepatocytes when total and CD8+ subset-enriched splenocytes were transferred. In contrast, we found only a few PCNA+ hepatocytes in mice transfused with CD4+ cells, where most of the PCNA+ cells were infiltrating inflammatory cells. Taken together, these results indicate that CD8+ T cells played a major role in induction of hepatocyte apoptosis and regenerative hepatocyte proliferation in this hepatitis model; CD4+ T cells did, however, play a minor role, especially in an early period of the acute phase.

Hepatocarcinogenesis Associated with Prolonged Chronic Liver Disease Induced by Differential Immune Cell Subsets.
We have reported that HCC development was primarily dependent on prolonged chronic inflammation in the liver after transfer of HBsAg-primed, total splenocytes (21) . To evaluate the relative procarcinogenic potential of liver disease induced by differential immune cell subsets, tumor development was monitored for 11–22 months in the mice transferred with the primed splenocyte subsets (Table 1)Citation . Twelve of the 14 animals transferred with CD8+ subset-enriched splenocytes developed HCC, the incidence of which was comparable with that seen after total cell transfer (11 of 12; Refs. 21 , 22 ). Most of them displayed multiple tumors, the size of the largest tumor was ranging up to 20 mm in diameter, and they illustrated the classical histological features of well-differentiated HCC including clear cells (Fig. 5, A and F)Citation . The surrounding hepatic parenchyma displayed focal lobular inflammatory infiltrates associated with degenerating hepatocytes, marked lobular disarray, fatty deposits, and clear tumor cell nests. The expression of HBsAg was abolished or decreased to a great extent in the tumor cells probably due to the altered transcription state (10 , 11) , whereas it was detectable in the surrounding parenchyma (Fig. 5, B and G)Citation . Even in the surrounding tissue the levels of expression were diminished, which was presumably due to long-term persistent inflammatory stimulation via noncytolytic cytokine-dependent mechanisms (36 , 37) . In addition, well-differentiated HCC consisting of tumor cells with large amounts of fatty deposits (Fig. 5C)Citation were seen in the same liver tissue of the mouse indicated in Fig. 5ACitation , suggesting a high potential for tumorigenesis induced by chronic immune-mediated liver disease. In tumor tissues of the mice transferred with CD8+ subset-enriched cells, we observed both highly differentiated, fat deposited neoplastic hepatocytes (Fig. 5HCitation , right area) and poorly differentiated, sarcomatous components (Fig. 5HCitation , left area). Furthermore, 2 of the mice were complicated with the rupture of HCC and bloody ascites (Fig. 5, I and J)Citation . All of the data indicate that prolonged chronic inflammation caused by the CD8+ subset displayed virtually the same effects as that by total splenocytes on the induction of hepatocarcinogenesis.


View this table:
[in this window]
[in a new window]

  		Table 1 Hepatocarcinogenesis after transfer of differential immune cell subsets

 


View larger version (104K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Histopathological features of progressive liver disease and hepatocellular carcinomas (HCC) development after transfer of total (A–C), CD4+ T cell-enriched (D and E) or CD8+ T cell-enriched (F–J) splenocytes in a transgenic mouse model of chronic hepatitis B. The mice described in the legend to Fig. 2Citation were killed >11 months after the transfer, and liver tissues were stained with H&E (A, C–F, H, and J) and for hepatitis B surface antigen (HBsAg; B and G). Tumors in mice transferred with total splenocytes display histological evidence of well-differentiated HCC (arrowheads; A). The surrounding hepatic parenchyma show focal lobular inflammatory infiltrates associated with degenerating hepatocytes and marked lobular disarray. HBsAg expression is abolished in the tumor cells (arrowheads), whereas it is detectable in the surrounding parenchyma (B). In the same liver tissue as in A, well-differentiated HCC is seen that consists of tumor cells with large amounts of fatty deposits (arrowheads; C). Animals that received CD4+-enriched cells demonstrate lower levels of inflammatory infiltrates (D) and clear tumor cell nests (arrowheads; E). Comparable with the mice that received total splenocyte transfer, animals that received CD8+-enriched cells show well-differentiated HCC including clear cells (arrowheads; F), the histological features of chronic liver disease in the surrounding hepatic parenchyma, and the suppression of HBsAg expression in the tumor cells (arrowheads; G). In tumor tissues, both highly differentiated, fat-deposited neoplastic hepatocytes (arrow; H, right area) and sarcomatous component (H, left area) were seen. Furthermore, the mice were complicated with the rupture of HCC (arrows; I and J). Original magnifications: x100 (A, D, E, F, and J), x40 (C), and x200 (B, G, and H). The bar represents 10 mm (I).

 
In contrast, the animals transferred with CD4+ subset-enriched splenocytes displayed a lower potential for hepatocarcinogenesis, in which 4 of the 17 mice developed HCC (Table 1)Citation . However, the tumors were histologically identical to those in the animals transferred with total splenocytes (Fig. 5, D and E)Citation . Interestingly, the numbers and sizes of tumors were comparable among the mice complicated with HCC after the transfer with total, CD4+, or CD8+ subset-enriched splenocytes [numbers of liver tumors/sizes (mm) in diameter of largest tumors (means ± SE); 4.2 ± 2.1/8.9 ± 1.7, 3.3 ± 1.3/7.8 ± 2.4, and 3.0 ± 0.7/10.2 ± 1.0, respectively; P > 0.5] (Table 1)Citation . The data indicate that the pathogenetic events induced by individual T-cell subsets influenced the potential in triggering the process of hepatocarcinogenesis rather than promoting tumor growth thereafter, resulting in different incidence of liver tumor. In addition, the mice thymectomized, irradiated, and transferred with splenocytes from the syngeneic transgenic animals or the unmanipulated controls developed no liver tumors. Collectively, the results demonstrate that each subset of the immune cells may individually contribute to the HCC development in this model. The CD8+ subset plays a primary role in the maintenance of chronic liver disease and induction of liver tumors, whereas the CD4+ subset displays a minimal potential for tumorigenesis during the process of prolonged chronic inflammation in the liver.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current study indicates that T cells, especially the CD8+ subset, rather than B cells are primarily responsible for the induction of prolonged liver injury in a mouse model of viral hepatitis. In contrast to the CD8+ T-cell subset, CD4+ T cells caused milder hepatic injury that seemed to improve shortly after the transfer as seen at the transaminase level. Consistent with the severity and duration of persistent liver disease, CD8+ T cells displayed strong induction of hepatocellular apoptosis, inflammation, and regenerative proliferation that sets up a cycle of liver cell destruction and regeneration. Furthermore, the pathogenetic events induced by individual T-cell subsets exerted their different potential in triggering the oncogenic process of hepatocarcinogenesis. On the basis of these results, we suggest that CD8+ T lymphocytes can principally contribute to the progression of chronic liver disease and the initiation of a complex sequence of events that leads to the development of HCC.

In the previous study, we observed that CD8+ CTL clones caused liver cell apoptosis by activating the FasL/Fas-, perforin/granzyme-, and cytokine-dependent death pathways in the model of acute hepatocellular injury (24) . Additionally, the liver cell injury was amplified by inflammation that may be exaggerated by FasL expressed on CD8+ CTL clones (18 , 19) . Similarly, the histological findings in the current study indicated that an intracellular caspase cascade, which is a death pathway of hepatocytes, was strongly activated after CD8+ T-cell transfer, and hepatocyte apoptosis was detectable along the area with inflammatory infiltrates. The massive cell loss through inflammation was thought to stimulate the regenerative proliferation of hepatocytes. These data suggest that the effector mechanism for chronic liver disease induced by CD8+ T cells may be similar to acute liver cell injury induced by the CTL clones.

In other mouse models, liver cell injury was induced by transfer of CD8+ CTL clones or hepatotoxic agents, and demonstrated acute, self-limited kinetics (17 , 18 , 30 , 38) . In the current experiment, prolonged immune-mediated hepatitis was established after transfer of CD8+ T cells obtained from HBsAg-primed, nontransgenic mice to the transgenic recipients. We speculated that the transferred, CD8+ T cells primarily contributed to the unique kinetics of liver disease because the CD8+ subset of splenocytes were prepared without an in vitro stimulation, indicating that not only effector but also memory T cells were included in the transplanted cells. CD8+ memory T cells were reported recently to have a longer life span than CD4+ memory T cells (39) . Thus, the lymphocytic choriomeningitis virus-specific CD8+ T cells appeared to survive in secondary lymphoid organs (periphery) and display their antiviral effects for >2 years (40, 41, 42) . Consistent with this finding, we observed previously that the HBsAg-specific, CD8+ CTL response was detected in the splenocytes 17 months after adoptive transfer in this chronic disease model (21) . Collectively, the transferred, memory CD8+ T cells are estimated to home to and survive in secondary lymphoid organs for >1 and 1.5 years, and suggested to continuously supply effector T cells into liver tissue through blood flow, which can recognize HBsAg expressed on the hepatocytes and maintain hepatocellular injury.

During the carcinogenetic process, it has been suggested that latent genetic mutations in the cells can be induced to undergo clonal selection in the initiation stage, and that the growth of the initiated cells that carry the first mutation can be stimulated in the promotion stage (43) . Accordingly, the oncogenic potential in the initiation and the promotion stages may be reflected by the numbers and sizes of tumors, respectively. In the current study, the transfer of the CD4+ and CD8+ T-cell subsets caused differences not in the numbers or sizes but in the incidences of HCC, suggesting that the pathogenetic events directly affected neither stage of carcinogenesis. We speculate that the events induced by the individual subsets might be involved in triggering carcinogenetic environment to different extents in the earlier stage than in the initiation. And, irrespective of the causative T-cell subsets, initiation and promotion of HCC would occur when such environment was generated. This sequence of events suggests that the regulation of T cell-induced environment in the early period of hepatocarcinogenesis may inhibit tumor cell initiation and cancel the following malignant transformation leading to tumor development.

The precise mechanisms of hepatocarcinogenesis during chronic viral hepatitis still remain unanswered. Within the entire spectrum of carcinogenic pathways including hepatic cell injury (13 , 44 , 45) , proliferation (46, 47, 48) , altered gene expression (49 , 50) , and the role of p53 (51, 52, 53, 54) , the current study design evaluating the late events in the carcinogenic process may not be sufficient to discuss the disease hypothesis. However, it is most likely, based on the striking similarity of the disease processes in human viral hepatitis and the animal model used in the current study (21 , 22) , that the T-cell subsets are critically involved in the pathogenesis of HCC associated with chronic hepatitis B. Elucidating the contribution of immunological events to the HCC development should be important not only for understanding the pathogenesis of liver cancer, but also for establishing new cancer-preventive treatments for patients with viral hepatitis.


   ACKNOWLEDGMENTS
 
We thank Dr. Francis V. Chisari for kindly providing us the HBsAg transgenic mice, and Akemi Nakano, Yoko Hashimoto, Maki Kawamura, and Chiharu Minami for technical assistance.


   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.

Requests for reprints: Shuichi Kaneko, Department of Gastroenterology; Graduate School of Medicine, Kanazawa University; 13-1 Takara-machi, Kanazawa 920-8641, Japan. Phone: 81-76-265-2231; Fax: 81-76-234-4250; E-mail: skaneko{at}medf.m.kanazawa-u.ac.jp

4 Unpublished observations. Back

Received 12/ 6/03. Revised 2/ 5/04. Accepted 2/25/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Di Bisceglie AM Hepatitis C and hepatocellular carcinoma. Hepatology, 26: 34S-8S, 1997.[CrossRef][Medline]
  2. Ikeda K, Saitoh S, Suzuki Y, et al Disease progression and hepatocellular carcinogenesis in patients with chronic viral hepatitis: a prospective observation of 2215 patients. J Hepatol, 28: 930-8, 1998.[CrossRef][Medline]
  3. Kim CM, Koike K, Saito I, Miyamura T, Jay G HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature, 351: 317-20, 1991.[CrossRef][Medline]
  4. Koike K, Moriya K, Iino S, et al High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology, 19: 810-9, 1994.[CrossRef][Medline]
  5. Moriya K, Fujie H, Shintani Y, et al The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med, 4: 1065-7, 1998.[CrossRef][Medline]
  6. Lerat H, Honda M, Beard MR, et al Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology, 122: 352-65, 2002.[CrossRef][Medline]
  7. Matsubara K, Tokino T Integration of hepatitis B virus DNA and its implications for hepatocarcinogenesis. Mol Biol Med, 7: 243-60, 1990.[Medline]
  8. Hildt E, Saher G, Bruss V, Hofschneider PH The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology, 225: 235-9, 1996.[CrossRef][Medline]
  9. Meyer M, Caselmann WH, Schluter V, Schreck R, Hofschneider PH, Baeuerle PA Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization. EMBO J, 11: 2991-3001, 1992.[Medline]
  10. Chisari FV, Klopchin K, Moriyama T, et al Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell, 59: 1145-56, 1989.[CrossRef][Medline]
  11. Dunsford HA, Sell S, Chisari FV Hepatocarcinogenesis due to chronic liver cell injury in hepatitis B virus transgenic mice. Cancer Res, 50: 3400-7, 1990.[Abstract/Free Full Text]
  12. Hagen TM, Huang S, Curnutte J, et al Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc Natl Acad Sci USA, 91: 12808-12, 1994.[Abstract/Free Full Text]
  13. Chisari FV, Ferrari C Hepatitis B virus immunopathogenesis. Annu Rev Immunol, 13: 29-60, 1995.[CrossRef][Medline]
  14. Cerny A, Chisari FV Immunological aspects of HCV infection. Intervirology, 37: 119-25, 1994.[Medline]
  15. Chang KM, Rehermann B, Chisari FV Immunopathology of hepatitis C. Springer Semin. Immunopathol, 19: 57-68, 1997.[CrossRef][Medline]
  16. Rehermann B, Chisari FV Cell mediated immune response to the hepatitis C virus. Curr Top Microbiol Immunol, 242: 299-325, 2000.[Medline]
  17. Moriyama T, Guilhot S, Klopchin K, et al Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science, 248: 361-4, 1990.[Abstract/Free Full Text]
  18. Ando K, Moriyama T, Guidotti LG, et al Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med, 178: 1541-54, 1993.[Abstract/Free Full Text]
  19. Ando K, Guidotti LG, Wirth S, et al Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J Immunol, 152: 3245-53, 1994.[Abstract]
  20. Franco A, Guidotti LG, Hobbs MV, Pasquetto V, Chisari FV Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice. J Immunol, 159: 2001-8, 1997.[Abstract]
  21. Nakamoto Y, Guidotti LG, Kuhlen CV, Fowler P, Chisari FV Immune pathogenesis of hepatocellular carcinoma. J Exp Med, 188: 341-50, 1998.[Abstract/Free Full Text]
  22. Nakamoto Y, Kaneko S, Fan H, et al Prevention of hepatocellular carcinoma development associated with chronic hepatitis by anti-fas ligand antibody therapy. J Exp Med, 196: 1105-11, 2002.[Abstract/Free Full Text]
  23. Kondo T, Suda T, Fukuyama H, Adachi M, Nagata S Essential roles of the Fas ligand in the development of hepatitis. Nat Med, 3: 409-13, 1997.[CrossRef][Medline]
  24. Nakamoto Y, Guidotti LG, Pasquetto V, Schreiber RD, Chisari FV Differential target cell sensitivity to CTL-activated death pathways in hepatitis B virus transgenic mice. J Immunol, 158: 5692-7, 1997.[Abstract]
  25. Suda T, Takahashi T, Golstein P, Nagata S Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell, 75: 1169-78, 1993.[CrossRef][Medline]
  26. Rouvier E, Luciani MF, Golstein P Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity. J Exp Med, 177: 195-200, 1993.[Abstract/Free Full Text]
  27. Anel A, Buferne M, Boyer C, Schmitt-Verhulst AM, Golstein P T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A. Eur J Immunol, 24: 2469-76, 1994.[Medline]
  28. Vignaux F, Vivier E, Malissen B, Depraetere V, Nagata S, Golstein P TCR/CD3 coupling to Fas-based cytotoxicity. J Exp Med, 181: 781-6, 1995.[Abstract/Free Full Text]
  29. Suda T, Okazaki T, Naito Y, et al Expression of the Fas ligand in cells of T cell lineage. J Immunol, 154: 3806-13, 1995.[Abstract]
  30. Chisari FV, Filippi P, McLachlan A, et al Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol, 60: 880-7, 1986.[Abstract/Free Full Text]
  31. Chisari FV, Filippi P, Buras J, et al Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Sci USA, 84: 6909-13, 1987.[Abstract/Free Full Text]
  32. Wirth S, Guidotti LG, Ando K, Schlicht HJ, Chisari FV Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus envelope transgenic mice. J Immunol, 154: 2504-15, 1995.[Abstract]
  33. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W . Current Protocols in Immunology, John Wiley & Sons, Inc. New York 1994.
  34. Guidotti LG, Martinez V, Loh YT, Rogler CE, Chisari FV Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J Virol, 68: 5469-75, 1994.[Abstract/Free Full Text]
  35. Urase K, Fujita E, Miho Y, et al Detection of activated caspase-3 (CPP32) in the vertebrate nervous system during development by a cleavage site-directed antiserum. Brain Res Dev Brain Res, 111: 77-87, 1998.[CrossRef][Medline]
  36. Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity, 4: 25-36, 1996.[CrossRef][Medline]
  37. Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV Viral clearance without destruction of infected cells during acute HBV infection. Science, 284: 825-9, 1999.[Abstract/Free Full Text]
  38. Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma. Hepatology, 16: 655-63, 1992.[Medline]
  39. Homann D, Teyton L, Oldstone MB Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat Med, 7: 913-9, 2001.[CrossRef][Medline]
  40. Lau LL, Jamieson BD, Somasundaram T, Ahmed R Cytotoxic T-cell memory without antigen. Nature, 369: 648-52, 1994.[CrossRef][Medline]
  41. Murali-Krishna K, Lau LL, Sambhara S, Lemonnier F, Altman J, Ahmed R Persistence of memory CD8 T cells in MHC class I-deficient mice. Science, 286: 1377-81, 1999.[Abstract/Free Full Text]
  42. Kaech SM, Wherry EJ, Ahmed R Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol, 2: 251-62, 2002.[CrossRef][Medline]
  43. Perez-Losada J, Balmain A Stem-cell hierarchy in skin cancer. Nat Rev Cancer, 3: 434-43, 2003.[CrossRef][Medline]
  44. Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med, 132: 296-305, 2000.[Abstract/Free Full Text]
  45. Nakamoto Y, Kaneko S Mechanisms of viral hepatitis induced liver injury. Curr Mol Med, 3: 537-44, 2003.[CrossRef][Medline]
  46. Columbano A, Shinozuka H Liver regeneration versus direct hyperplasia. FASEB J, 10: 1118-28, 1996.[Abstract]
  47. Sell S Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology, 33: 738-50, 2001.[CrossRef][Medline]
  48. Coleman WB Mechanisms of human hepatocarcinogenesis. Curr Mol Med, 3: 573-88, 2003.[CrossRef][Medline]
  49. Shirota Y, Kaneko S, Honda M, Kawai HF, Kobayashi K Identification of differentially expressed genes in hepatocellular carcinoma with cDNA microarrays. Hepatology, 33: 832-40, 2001.[CrossRef][Medline]
  50. Xu XR, Huang J, Xu ZG, et al Insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver. Proc Natl Acad Sci USA, 98: 15089-94, 2001.[Abstract/Free Full Text]
  51. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350: 427-8, 1991.[CrossRef][Medline]
  52. Bressac B, Kew M, Wands J, Ozturk M Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature, 350: 429-31, 1991.[CrossRef][Medline]
  53. Kern MA, Breuhahn K, Schirmacher P Molecular pathogenesis of human hepatocellular carcinoma. Adv Cancer Res, 86: 67-112, 2002.[Medline]
  54. Eferl R, Ricci R, Kenner L, et al Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell, 112: 181-92, 2003.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Virol.Home page
B. S. Sheridan, T. L. Cherpes, J. Urban, P. Kalinski, and R. L. Hendricks
Reevaluating the CD8 T-Cell Response to Herpes Simplex Virus Type 1: Involvement of CD8 T Cells Reactive to Subdominant Epitopes
J. Virol., March 1, 2009; 83(5): 2237 - 2245.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. L. Cherpes, J. L. Busch, B. S. Sheridan, S. A. K. Harvey, and R. L. Hendricks
Medroxyprogesterone Acetate Inhibits CD8+ T Cell Viral-Specific Effector Function and Induces Herpes Simplex Virus Type 1 Reactivation
J. Immunol., July 15, 2008; 181(2): 969 - 975.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
N. Kobayashi, N. Hiraoka, W. Yamagami, H. Ojima, Y. Kanai, T. Kosuge, A. Nakajima, and S. Hirohashi
FOXP3+ Regulatory T Cells Affect the Development and Progression of Hepatocarcinogenesis
Clin. Cancer Res., February 1, 2007; 13(3): 902 - 911.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kaneko, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakamoto, Y.
Right arrow Articles by Kaneko, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online