Contribution of the Lymphotoxin Receptor to Liver Regeneration1
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免疫学杂志 2005年第14期
Abstract
The liver has an enormous capacity to regenerate in response to insults, but the cellular events and molecules involved in liver regeneration are not well defined. In this study, we report that ligands expressed on the surface of lymphocytes have a substantial effect on liver homeostasis. We demonstrate that a T cell-restricted ligand, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator on T cells (LIGHT), signaling through the lymphotoxin receptor (LTR) expressed on mature hepatocytes induces massive hepatomegaly. Using genetic targeting and a receptor fusion protein, we further show that mice deficient in LTR signaling have a severe defect in their ability to survive partial hepatectomy with marked liver damage and failure to initiate DNA synthesis after partial hepatectomy. We further show that mice deficient in a LTR ligand, LT, also show decreased ability to survive partial hepatectomy with similar levels of liver damage and decreased DNA synthesis. Therefore, our study has revealed an unexpected role of lymphocyte-restricted ligands and defined a new pathway in supporting liver regeneration.
Introduction
The liver is a vital organ that performs many biological functions including synthesis, metabolism, and storage of carbohydrates, proteins, and lipids. The hepatocytes, which comprise the dominant cell population of the liver, effect these functions. Survival from insults such as viruses and toxins requires maintenance of hepatic mass. Therefore, in addition to the synthetic and metabolic function, the liver must continue to undergo self-renewal (1). Controlling the ability of the liver to regulate its mass has broad clinical implications. Organ shortages continue to press the need for liver transplantation alternatives, and the ability to increase the renewal of the liver would make split liver transplants and cellular transplants more feasible. In addition, flares of necroinflammatory activity seen in chronic hepatitis lead to increased hepatocyte turnover and fibrosis. Further understanding of liver growth control mechanisms offers a possible solution to both liver transplantation and chronic liver disease.
The partial hepatectomy is a particularly useful model for beginning to understand how the liver controls self-renewal (2). Partial hepatectomy, a procedure that removes 70% of the liver, is regarded as the preferred in vivo method to study liver growth due to its synchronized growth response in the liver (3, 4). The understanding of liver regeneration has focused on the cytokine and growth factor network involved in the process (5). These studies have defined TNF- and IL-6 as important cytokines that prime the hepatocyte for response to growth factors such as hepatocyte growth factor, epidermal growth factor, and TGF- (6, 7).
Besides release of cytokines and growth factors after partial hepatectomy, rapid changes in the cellular composition of the liver occur immediately after partial hepatectomy. For example, as early as 12 h after partial hepatectomy, there is an increase in the number of intrahepatic lymphocytes. This increase is due to an increase in the percentage of CD4-positive and NK T cells (8). However, the contribution of lymphocytes to the regenerative process has not been defined.
The lymphocyte-derived ligand, homologous to lymphotoxin (LT),3 exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells (LIGHT) is a TNF family member (TNF superfamily 14) that is expressed on activated T cells (9). LIGHT has two receptors, the LTR, which is expressed on nonhemopoietic cells, and the herpes virus entry mediator receptor (HVEM), which is expressed on hemopoietic cells (9, 10). The expression of LIGHT on activated T cells can signal to lymphocytes via the HVEM receptor and to nonhemopoietic cells through the LTR. Therefore, LIGHT performs a unique role in bridging hemopoietic and nonhemopoietic signaling.
In this study, we show that transgenic mice expressing LIGHT (Tg LIGHT) under the control of the T cell-restricted p56lck promoter and CD2 enhancer display a massive increase in liver mass and markedly abnormal hepatocyte histology. The effect of LIGHT is dependent on the LTR, which we demonstrate is expressed on hepatocytes. Immediately after partial hepatectomy, there is an increase in LIGHT and LT from intrahepatic lymphocytes. Furthermore, LTR-deficient mice (LTR–/–) show an impaired ability to regenerate their liver after partial hepatectomy, suggesting that the LTR pathway is required for optimal liver regeneration. We demonstrate the importance of the LTR signaling in supporting liver biology by demonstrating a deficiency in LT also shows an impaired ability to undergo liver regeneration. Finally, we are able to show that treatment with a LTR human fusion protein (LTR-hFc) also blocks liver regeneration in wild-type mice. Therefore, this study establishes a key interaction between lymphocyte-derived ligands and hepatocytes and uncovers the LTR and its ligands as a new pathway during liver regeneration.
Materials and Methods
Mice and reagents
Mice carrying a transgene for LIGHT have previously been reported (11). Male C57B/L6J, LT–/– mice (6–10 wk old) were a gift from D. Chaplin (Washington University, St. Louis, MO) and were backcrossed to a B6 background for 13 generations. LTR–/– mice were generated by K. Pfeffer (Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany) (12) and backcrossed to a B6 background for seven generations. HVEM–/– mice were recently generated in K. Pfeffer’s laboratory and backcrossed to B6 for five generations with genetic targeting confirmed by Southern blot analysis and Ab staining (13). All mice were maintained at the University of Chicago and handled according to National Institutes of Health guideline and approved by the Institutional Animal Care and Use Committee. Bone marrow transplantation was performed by lethally irradiating recipients with 900 rad followed by i.v. injection of 3 million bone marrow cells which were isolated by flushing the femur and humerus of the donor with sterile PBS. Adoptive transfer was performed by harvesting the thymus from 6-wk-old mice and i.v. injection of 20 x 106 cells into RAG1–/– mice (The Jackson Laboratory). Treatment with LTR-hFc (Biogen), was performed by i.v. injection of 200 μg of fusion protein 24 h before and immediately after partial hepatectomy. Anti-LTR (ACH6) and negative control (Ha4/8) hamster Ab were kindly provided by J. Browning (Biogen).
Partial hepatectomy
The procedure was performed as originally described by Higgins and Anderson (4). The mice were anesthetized with ketamine and xylazine (100 mg/kg/10 mg/kg i.p.), the liver was exposed through a midline incision, and the median and left lobes were mobilized and delivered after ligature with a single silk 4-0 suture. Sham procedures consist of anesthetizing mice followed by opening of the skin and peritoneum and manipulating but not resecting the liver. Serum transaminase activity was determined by the method of Karmen using an Elan Diagnostics analyzer according to the manufacturer’s protocols in the University of Chicago Animal Research Core laboratory.
Fluorescent staining
Frozen sections were cut from livers, allowed to air dry for 15 min, and incubated in PBS, 0.2% saponin, 5% normal goat serum for 15 min. Primary anti-LTR Ab (clone ACH6) or control primary Ab (Ha 4/8), 10 μg/ml in 1% normal goat serum in PBS, was incubated on the tissue in a humid chamber for 2 h at room temperature. Secondary Ab consisted of goat anti-hamster IgG Alex 594 (Molecular Probes), 10 μg/ml, and Alexa 488 phalloidin (Molecular Probes) in 1% normal goat serum in PBS for 1 h at room temperature.
Histology, BrdU, and TUNEL labeling
Tissue sections were fixed in 70% alcohol for 2 h followed by buffered formalin and processed either for routine H&E staining or for BrdU and immunohistochemical studies as per the manufacturer’s instructions (Zymed Laboratories). TUNEL staining was performed on paraffin-embedded, formalin-fixed tissue using the ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Chemicon International) according to the manufacturer’s directions. BrdU incorporation and TUNEL-positive nuclei were scored in 20 nonoverlapping high power fields (x40).
Results
Increase in T cell-restricted ligand, LIGHT, leads to hepatomegaly in a LTR-dependent manner
The liver regulates its mass through a strict homeostatic mechanism which maintains a 4–8% liver-body weight ratio (1). Much to our surprise, a TNF superfamily ligand, LIGHT (TNSF14), expressed under the proximal p56lck promoter and CD2 enhancer, has a substantially increased liver size compared with littermate wild-type control mice (Fig. 1A). When expressed as a percentage of total body weight, Tg LIGHT mice have significantly higher liver-body weigh ratios (Fig. 1B). Liver histology reveals markedly enlarged hepatocytes and frequent mitotic figures in Tg LIGHT mice compared with age matched wild-type livers (Fig. 1, C and D). The hepatomegaly, abnormal histology, and frequent mitotic figures in Tg LIGHT mice raise the possibility that a T cell-derived cytokine disrupts the mechanisms that normally balance hepatocyte proliferation and death.
These data led us to consider whether decreased survival in the absence of LTR signaling had a direct effect on the liver. LTR–/– and LT–/– mice showed evidence of liver damage 48 h after partial hepatectomy with significantly elevated serum aminotransferase levels compared with similarly treated wild-type mice (Fig. 6A). Histological examination of the livers showed larger areas of necrosis in the LTR–/– and LT–/– mice 48 h after partial hepatectomy compared with wild-type mice (Fig. 6B).
We also investigated whether there is an increase in the number of apoptotic hepatocytes using TUNEL staining. We could clearly demonstrate the number of TUNEL-positive nuclei were significantly increased in LTR–/– compared with wild-type mice at 24 and 48 h after partial hepatectomy (Fig. 6C). Evidence that LTR–/– mice develop extensive areas of hepatocyte death when challenged with partial hepatectomy is consistent with an in vitro analysis that the LTR-mediated microenvironment transduces viability signals in hepatocytes (14).
LTR signaling is essential in initiating DNA synthesis after partial hepatectomy
Next, we wanted to determine whether LTR signaling was involved in initiating DNA synthesis after partial hepatectomy. Mice received a 2-h pulse with a thymidine analog, BrdU, 48 h after partial hepatectomy or a sham procedure. This allowed us to carefully examine the proliferation of the hepatocyte after partial hepatectomy by counting the number of BrdU-positive hepatocyte nuclei after immunohistochemical staining. There was significantly more DNA synthesis in the wild-type mice than in either LTR–/– or LT–/– mice (Fig. 7A). As expected, sham procedures did not induce DNA synthesis.
The inability of the LTR–/– mice to synthesize DNA after partial hepatectomy was not due to a developmental lesion because short term treatment with LTR-hFc also blocked the initiation of DNA synthesis in wild-type mice (Fig. 7B). This indicated that signaling through the LTR as well as a LTR ligand(s) was important for the ability of the liver to regenerate hepatic mass. The combination of increased liver damage and a reduced regenerative response after partial hepatectomy induces significant mortality in mice that cannot activate the LTR. Together, these data suggest that LTR signaling through T cell-restricted ligands is critical for liver homeostasis.
Discussion
In this study, we have revealed for the first time that signaling through the LTR can have a substantial effect on the liver and that lymphocytes can play a novel role in liver regeneration. Our initial observation of proliferative and massively enlarged livers in Tg LIGHT mice led us to investigate two major questions. We wanted to know 1) whether a T cell-restricted ligand could have a dramatic liver phenotype and 2) what effect the ligand and receptor pathway has on the liver after partial hepatectomy.
We have previously demonstrated the expression of LIGHT on the T cells of Tg LIGHT mice; this agrees with what is known about the p56lck promoter and CD2 enhancer that drives the LIGHT transgene (11, 15, 16, 17). Tg LIGHT mice have marked T cell infiltration of peripheral organs. Because there was a correlation between the presence of increased LIGHT expressing T cells and enlargement of the liver, we sought to determine whether this was a direct or indirect effect. In support of the direct action of T cell-derived LIGHT, we have found that either bone marrow transplantation or transfer of thymocytes from Tg LIGHT mice into wild-type or RAG1–/– mice recipients resulted in the transfer of the abnormal liver phenotype. In addition, the direct effect of LIGHT on the liver was dependent on LTR expressed on mature hepatocytes, but not on the HVEM receptor. This suggests that LIGHT was directly mediating effects on the liver. Therefore, based on our observations, it appears as though the LTR functions as a bridge between LIGHT-expressing T cells and LTR expression on hepatocytes.
In the Tg LIGHT mice, there exists a strong likelihood of a direct interaction between the T cell and the hepatocyte. The Tg LIGHT mice were engineered to retain LIGHT on the surface of the T cell through the introduction of a mutation at a suspected cleavage site. In the nontransgenic system, LIGHT is expressed on activated T cells, with few other cell types expressing LIGHT. It is possible that activated T cells deliver LIGHT to the liver and that LIGHT is then cleaved from the T cell membrane. Soluble LIGHT could then interact with the LTR expressed on hepatocytes. The latter scenario is analogous to what occurs with other TNF superfamily ligands (18).
The physiological role of LIGHT and LTR in liver has not been defined. The increased expression of LIGHT in our Tg mice was used to hint at LIGHT’s potential role during inflammation. There is in vitro evidence to suggest LIGHT does have a direct role on hepatocytes. Treatment of cultured human hepatocytes with LIGHT protects against TNF-mediated apoptosis (14). LIGHT and the LTR does not appear to be essential for liver development during embryogenesis because mice deficient in LIGHT or LTR appear to have normal liver histology (data not shown).
LTR signaling by LIGHT appeared to mediate a proliferative environment in the liver, so we sought to define the role of the LTR in liver growth. Partial hepatectomy is an in vivo method to deliver a strong growth stimulus to hepatocytes. First, we were able to show an increase in the mRNA of lymphocyte-derived LTR ligands, LT and LIGHT, immediately after partial hepatectomy. Second, a deficiency in the LTR had a profound effect on the liver following partial hepatectomy. Not only was there a reduction in hepatocyte DNA synthesis but also there was significant liver damage and high mortality after partial hepatectomy. Third, the impaired liver-regenerative response could be replicated in wild-type mice treated with LTR-Fc, suggesting a direct role for the LTR. The LTR appears to be uniquely situated to balance cellular death and DNA synthesis in hepatocytes after partial hepatectomy. LTR signaling and the relative contribution of LIGHT and LT to normal liver cell turnover will have to be explored.
With this study, we have been able to uncover an unexpected role of lymphocyte-restricted ligands and defined a new pathway in supporting liver regeneration. This system will allow us to 1) explore the environment created by activated lymphocytes during liver regeneration, 2) explore the events downstream of the LTR that either protect hepatocytes from cell death, or 3) promote proliferation after partial hepatectomy.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by National Institutes of Health Grants R01-DK58897 and AI062026. R.A.A. was in part supported by a National Institutes of Health Training Grant (AI07090) and K08 DK067187.
2 Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of Pathology, University of Chicago, Chicago, IL 60637. E-mail address: yfu@uchicago.edu
3 Abbreviations used in this paper: LT, lymphotoxin; LIGHT, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator on T cells, a receptor expressed on T cells; HVEM, herpes virus entry mediator; Tg, transgenic; RAG1, recombinase-activating gene 1; LTR-hFc, LTR human fusion protein.
Received for publication February 25, 2005. Accepted for publication April 30, 2005.
References
Michalopoulos, G. K., M. C. DeFrances. 1997. Liver regeneration. Science 276: 60-66.
Diehl, A. M.. 2002. Liver regeneration. Front Biosci. 7: e301-e314.
Fausto, N., J. S. Campbell. 2003. The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech. Dev. 120: 117-122.
Higgins, G. M., R. M. Anderson. 1931. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12: 186-202.
Galun, E., J. H. Axelrod. 2002. The role of cytokines in liver failure and regeneration: potential new molecular therapies. Biochim. Biophys. Acta 1592: 345-358.
Cressman, D. E., L. E. Greenbaum, R. A. DeAngelis, G. Ciliberto, E. E. Furth, V. Poli, R. Taub. 1996. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274: 1379-1383.
Akerman, P., P. Cote, S. Q. Yang, C. McClain, S. Nelson, G. J. Bagby, A. M. Diehl. 1992. Antibodies to tumor necrosis factor- inhibit liver regeneration after partial hepatectomy. Am. J. Physiol. 263: G579-G585.
imizu, M. Bannai, H. Kawamura, K. Hatakeyama, T. Abo. 2000. Intensive expansion of natural killer T cells in the early phase of hepatocyte regeneration after partial hepatectomy in mice and its association with sympathetic nerve activation. Hepatology 31: 907-915.
The liver has an enormous capacity to regenerate in response to insults, but the cellular events and molecules involved in liver regeneration are not well defined. In this study, we report that ligands expressed on the surface of lymphocytes have a substantial effect on liver homeostasis. We demonstrate that a T cell-restricted ligand, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator on T cells (LIGHT), signaling through the lymphotoxin receptor (LTR) expressed on mature hepatocytes induces massive hepatomegaly. Using genetic targeting and a receptor fusion protein, we further show that mice deficient in LTR signaling have a severe defect in their ability to survive partial hepatectomy with marked liver damage and failure to initiate DNA synthesis after partial hepatectomy. We further show that mice deficient in a LTR ligand, LT, also show decreased ability to survive partial hepatectomy with similar levels of liver damage and decreased DNA synthesis. Therefore, our study has revealed an unexpected role of lymphocyte-restricted ligands and defined a new pathway in supporting liver regeneration.
Introduction
The liver is a vital organ that performs many biological functions including synthesis, metabolism, and storage of carbohydrates, proteins, and lipids. The hepatocytes, which comprise the dominant cell population of the liver, effect these functions. Survival from insults such as viruses and toxins requires maintenance of hepatic mass. Therefore, in addition to the synthetic and metabolic function, the liver must continue to undergo self-renewal (1). Controlling the ability of the liver to regulate its mass has broad clinical implications. Organ shortages continue to press the need for liver transplantation alternatives, and the ability to increase the renewal of the liver would make split liver transplants and cellular transplants more feasible. In addition, flares of necroinflammatory activity seen in chronic hepatitis lead to increased hepatocyte turnover and fibrosis. Further understanding of liver growth control mechanisms offers a possible solution to both liver transplantation and chronic liver disease.
The partial hepatectomy is a particularly useful model for beginning to understand how the liver controls self-renewal (2). Partial hepatectomy, a procedure that removes 70% of the liver, is regarded as the preferred in vivo method to study liver growth due to its synchronized growth response in the liver (3, 4). The understanding of liver regeneration has focused on the cytokine and growth factor network involved in the process (5). These studies have defined TNF- and IL-6 as important cytokines that prime the hepatocyte for response to growth factors such as hepatocyte growth factor, epidermal growth factor, and TGF- (6, 7).
Besides release of cytokines and growth factors after partial hepatectomy, rapid changes in the cellular composition of the liver occur immediately after partial hepatectomy. For example, as early as 12 h after partial hepatectomy, there is an increase in the number of intrahepatic lymphocytes. This increase is due to an increase in the percentage of CD4-positive and NK T cells (8). However, the contribution of lymphocytes to the regenerative process has not been defined.
The lymphocyte-derived ligand, homologous to lymphotoxin (LT),3 exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells (LIGHT) is a TNF family member (TNF superfamily 14) that is expressed on activated T cells (9). LIGHT has two receptors, the LTR, which is expressed on nonhemopoietic cells, and the herpes virus entry mediator receptor (HVEM), which is expressed on hemopoietic cells (9, 10). The expression of LIGHT on activated T cells can signal to lymphocytes via the HVEM receptor and to nonhemopoietic cells through the LTR. Therefore, LIGHT performs a unique role in bridging hemopoietic and nonhemopoietic signaling.
In this study, we show that transgenic mice expressing LIGHT (Tg LIGHT) under the control of the T cell-restricted p56lck promoter and CD2 enhancer display a massive increase in liver mass and markedly abnormal hepatocyte histology. The effect of LIGHT is dependent on the LTR, which we demonstrate is expressed on hepatocytes. Immediately after partial hepatectomy, there is an increase in LIGHT and LT from intrahepatic lymphocytes. Furthermore, LTR-deficient mice (LTR–/–) show an impaired ability to regenerate their liver after partial hepatectomy, suggesting that the LTR pathway is required for optimal liver regeneration. We demonstrate the importance of the LTR signaling in supporting liver biology by demonstrating a deficiency in LT also shows an impaired ability to undergo liver regeneration. Finally, we are able to show that treatment with a LTR human fusion protein (LTR-hFc) also blocks liver regeneration in wild-type mice. Therefore, this study establishes a key interaction between lymphocyte-derived ligands and hepatocytes and uncovers the LTR and its ligands as a new pathway during liver regeneration.
Materials and Methods
Mice and reagents
Mice carrying a transgene for LIGHT have previously been reported (11). Male C57B/L6J, LT–/– mice (6–10 wk old) were a gift from D. Chaplin (Washington University, St. Louis, MO) and were backcrossed to a B6 background for 13 generations. LTR–/– mice were generated by K. Pfeffer (Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany) (12) and backcrossed to a B6 background for seven generations. HVEM–/– mice were recently generated in K. Pfeffer’s laboratory and backcrossed to B6 for five generations with genetic targeting confirmed by Southern blot analysis and Ab staining (13). All mice were maintained at the University of Chicago and handled according to National Institutes of Health guideline and approved by the Institutional Animal Care and Use Committee. Bone marrow transplantation was performed by lethally irradiating recipients with 900 rad followed by i.v. injection of 3 million bone marrow cells which were isolated by flushing the femur and humerus of the donor with sterile PBS. Adoptive transfer was performed by harvesting the thymus from 6-wk-old mice and i.v. injection of 20 x 106 cells into RAG1–/– mice (The Jackson Laboratory). Treatment with LTR-hFc (Biogen), was performed by i.v. injection of 200 μg of fusion protein 24 h before and immediately after partial hepatectomy. Anti-LTR (ACH6) and negative control (Ha4/8) hamster Ab were kindly provided by J. Browning (Biogen).
Partial hepatectomy
The procedure was performed as originally described by Higgins and Anderson (4). The mice were anesthetized with ketamine and xylazine (100 mg/kg/10 mg/kg i.p.), the liver was exposed through a midline incision, and the median and left lobes were mobilized and delivered after ligature with a single silk 4-0 suture. Sham procedures consist of anesthetizing mice followed by opening of the skin and peritoneum and manipulating but not resecting the liver. Serum transaminase activity was determined by the method of Karmen using an Elan Diagnostics analyzer according to the manufacturer’s protocols in the University of Chicago Animal Research Core laboratory.
Fluorescent staining
Frozen sections were cut from livers, allowed to air dry for 15 min, and incubated in PBS, 0.2% saponin, 5% normal goat serum for 15 min. Primary anti-LTR Ab (clone ACH6) or control primary Ab (Ha 4/8), 10 μg/ml in 1% normal goat serum in PBS, was incubated on the tissue in a humid chamber for 2 h at room temperature. Secondary Ab consisted of goat anti-hamster IgG Alex 594 (Molecular Probes), 10 μg/ml, and Alexa 488 phalloidin (Molecular Probes) in 1% normal goat serum in PBS for 1 h at room temperature.
Histology, BrdU, and TUNEL labeling
Tissue sections were fixed in 70% alcohol for 2 h followed by buffered formalin and processed either for routine H&E staining or for BrdU and immunohistochemical studies as per the manufacturer’s instructions (Zymed Laboratories). TUNEL staining was performed on paraffin-embedded, formalin-fixed tissue using the ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Chemicon International) according to the manufacturer’s directions. BrdU incorporation and TUNEL-positive nuclei were scored in 20 nonoverlapping high power fields (x40).
Results
Increase in T cell-restricted ligand, LIGHT, leads to hepatomegaly in a LTR-dependent manner
The liver regulates its mass through a strict homeostatic mechanism which maintains a 4–8% liver-body weight ratio (1). Much to our surprise, a TNF superfamily ligand, LIGHT (TNSF14), expressed under the proximal p56lck promoter and CD2 enhancer, has a substantially increased liver size compared with littermate wild-type control mice (Fig. 1A). When expressed as a percentage of total body weight, Tg LIGHT mice have significantly higher liver-body weigh ratios (Fig. 1B). Liver histology reveals markedly enlarged hepatocytes and frequent mitotic figures in Tg LIGHT mice compared with age matched wild-type livers (Fig. 1, C and D). The hepatomegaly, abnormal histology, and frequent mitotic figures in Tg LIGHT mice raise the possibility that a T cell-derived cytokine disrupts the mechanisms that normally balance hepatocyte proliferation and death.
These data led us to consider whether decreased survival in the absence of LTR signaling had a direct effect on the liver. LTR–/– and LT–/– mice showed evidence of liver damage 48 h after partial hepatectomy with significantly elevated serum aminotransferase levels compared with similarly treated wild-type mice (Fig. 6A). Histological examination of the livers showed larger areas of necrosis in the LTR–/– and LT–/– mice 48 h after partial hepatectomy compared with wild-type mice (Fig. 6B).
We also investigated whether there is an increase in the number of apoptotic hepatocytes using TUNEL staining. We could clearly demonstrate the number of TUNEL-positive nuclei were significantly increased in LTR–/– compared with wild-type mice at 24 and 48 h after partial hepatectomy (Fig. 6C). Evidence that LTR–/– mice develop extensive areas of hepatocyte death when challenged with partial hepatectomy is consistent with an in vitro analysis that the LTR-mediated microenvironment transduces viability signals in hepatocytes (14).
LTR signaling is essential in initiating DNA synthesis after partial hepatectomy
Next, we wanted to determine whether LTR signaling was involved in initiating DNA synthesis after partial hepatectomy. Mice received a 2-h pulse with a thymidine analog, BrdU, 48 h after partial hepatectomy or a sham procedure. This allowed us to carefully examine the proliferation of the hepatocyte after partial hepatectomy by counting the number of BrdU-positive hepatocyte nuclei after immunohistochemical staining. There was significantly more DNA synthesis in the wild-type mice than in either LTR–/– or LT–/– mice (Fig. 7A). As expected, sham procedures did not induce DNA synthesis.
The inability of the LTR–/– mice to synthesize DNA after partial hepatectomy was not due to a developmental lesion because short term treatment with LTR-hFc also blocked the initiation of DNA synthesis in wild-type mice (Fig. 7B). This indicated that signaling through the LTR as well as a LTR ligand(s) was important for the ability of the liver to regenerate hepatic mass. The combination of increased liver damage and a reduced regenerative response after partial hepatectomy induces significant mortality in mice that cannot activate the LTR. Together, these data suggest that LTR signaling through T cell-restricted ligands is critical for liver homeostasis.
Discussion
In this study, we have revealed for the first time that signaling through the LTR can have a substantial effect on the liver and that lymphocytes can play a novel role in liver regeneration. Our initial observation of proliferative and massively enlarged livers in Tg LIGHT mice led us to investigate two major questions. We wanted to know 1) whether a T cell-restricted ligand could have a dramatic liver phenotype and 2) what effect the ligand and receptor pathway has on the liver after partial hepatectomy.
We have previously demonstrated the expression of LIGHT on the T cells of Tg LIGHT mice; this agrees with what is known about the p56lck promoter and CD2 enhancer that drives the LIGHT transgene (11, 15, 16, 17). Tg LIGHT mice have marked T cell infiltration of peripheral organs. Because there was a correlation between the presence of increased LIGHT expressing T cells and enlargement of the liver, we sought to determine whether this was a direct or indirect effect. In support of the direct action of T cell-derived LIGHT, we have found that either bone marrow transplantation or transfer of thymocytes from Tg LIGHT mice into wild-type or RAG1–/– mice recipients resulted in the transfer of the abnormal liver phenotype. In addition, the direct effect of LIGHT on the liver was dependent on LTR expressed on mature hepatocytes, but not on the HVEM receptor. This suggests that LIGHT was directly mediating effects on the liver. Therefore, based on our observations, it appears as though the LTR functions as a bridge between LIGHT-expressing T cells and LTR expression on hepatocytes.
In the Tg LIGHT mice, there exists a strong likelihood of a direct interaction between the T cell and the hepatocyte. The Tg LIGHT mice were engineered to retain LIGHT on the surface of the T cell through the introduction of a mutation at a suspected cleavage site. In the nontransgenic system, LIGHT is expressed on activated T cells, with few other cell types expressing LIGHT. It is possible that activated T cells deliver LIGHT to the liver and that LIGHT is then cleaved from the T cell membrane. Soluble LIGHT could then interact with the LTR expressed on hepatocytes. The latter scenario is analogous to what occurs with other TNF superfamily ligands (18).
The physiological role of LIGHT and LTR in liver has not been defined. The increased expression of LIGHT in our Tg mice was used to hint at LIGHT’s potential role during inflammation. There is in vitro evidence to suggest LIGHT does have a direct role on hepatocytes. Treatment of cultured human hepatocytes with LIGHT protects against TNF-mediated apoptosis (14). LIGHT and the LTR does not appear to be essential for liver development during embryogenesis because mice deficient in LIGHT or LTR appear to have normal liver histology (data not shown).
LTR signaling by LIGHT appeared to mediate a proliferative environment in the liver, so we sought to define the role of the LTR in liver growth. Partial hepatectomy is an in vivo method to deliver a strong growth stimulus to hepatocytes. First, we were able to show an increase in the mRNA of lymphocyte-derived LTR ligands, LT and LIGHT, immediately after partial hepatectomy. Second, a deficiency in the LTR had a profound effect on the liver following partial hepatectomy. Not only was there a reduction in hepatocyte DNA synthesis but also there was significant liver damage and high mortality after partial hepatectomy. Third, the impaired liver-regenerative response could be replicated in wild-type mice treated with LTR-Fc, suggesting a direct role for the LTR. The LTR appears to be uniquely situated to balance cellular death and DNA synthesis in hepatocytes after partial hepatectomy. LTR signaling and the relative contribution of LIGHT and LT to normal liver cell turnover will have to be explored.
With this study, we have been able to uncover an unexpected role of lymphocyte-restricted ligands and defined a new pathway in supporting liver regeneration. This system will allow us to 1) explore the environment created by activated lymphocytes during liver regeneration, 2) explore the events downstream of the LTR that either protect hepatocytes from cell death, or 3) promote proliferation after partial hepatectomy.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by National Institutes of Health Grants R01-DK58897 and AI062026. R.A.A. was in part supported by a National Institutes of Health Training Grant (AI07090) and K08 DK067187.
2 Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of Pathology, University of Chicago, Chicago, IL 60637. E-mail address: yfu@uchicago.edu
3 Abbreviations used in this paper: LT, lymphotoxin; LIGHT, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator on T cells, a receptor expressed on T cells; HVEM, herpes virus entry mediator; Tg, transgenic; RAG1, recombinase-activating gene 1; LTR-hFc, LTR human fusion protein.
Received for publication February 25, 2005. Accepted for publication April 30, 2005.
References
Michalopoulos, G. K., M. C. DeFrances. 1997. Liver regeneration. Science 276: 60-66.
Diehl, A. M.. 2002. Liver regeneration. Front Biosci. 7: e301-e314.
Fausto, N., J. S. Campbell. 2003. The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech. Dev. 120: 117-122.
Higgins, G. M., R. M. Anderson. 1931. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12: 186-202.
Galun, E., J. H. Axelrod. 2002. The role of cytokines in liver failure and regeneration: potential new molecular therapies. Biochim. Biophys. Acta 1592: 345-358.
Cressman, D. E., L. E. Greenbaum, R. A. DeAngelis, G. Ciliberto, E. E. Furth, V. Poli, R. Taub. 1996. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274: 1379-1383.
Akerman, P., P. Cote, S. Q. Yang, C. McClain, S. Nelson, G. J. Bagby, A. M. Diehl. 1992. Antibodies to tumor necrosis factor- inhibit liver regeneration after partial hepatectomy. Am. J. Physiol. 263: G579-G585.
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