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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0537fje. |
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Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Germany
3Correspondence: Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl Neubergstrasse 1, 30625 Hannover, Germany. E-mail: Kubicka.stefan{at}mh-hannover.de
SPECIFIC AIM
TRAIL is able to kill a broad spectrum of tumor cells, but appears to be nontoxic to most normal cells. Since there are conflicting data about hepatotoxicity of TRAIL, we investigated the physiological function of TRAIL and its receptors in the liver.
PRINCIPAL FINDINGS
TRAIL and FasL (CD95L) induce apoptosis of hepatocytes in vitro
We investigated the effects of membrane-bound FasL (CD95L) and TRAIL in cell lines and hepatocytes in vitro by adenovirus-mediated gene transfer. The Huh7 hepatoma cell line and primary cultured embryonal murine hepatocytes were resistant to Ad-FasL (CD95L) and Ad-TRAIL treatment as shown by the unchanged maintenance of their nuclear morphology in DAPI staining. Adult human and murine primary hepatocytes were sensitive for Ad-FasL (CD95L) and Ad-TRAIL-mediated apoptosis. However, murine hepatocytes seem to be more sensitive to Ad-FasL (CD95L) than to Ad-TRAIL-mediated apoptosis, in contrast to human hepatocytes.
In contrast to FasL, TRAIL-mediated apoptosis of hepatocytes in vivo needs triggering through virus infection
Having shown that murine hepatocytes are sensitive to membrane-bound TRAIL-mediated apoptosis in vitro, we determined whether TRAIL expression in the liver of mice in vivo could result in hepatotoxicity. Mice were killed 24 h after infection with 1 x 107 pfu/g of adenoviral vectors and the livers were harvested for TUNEL assays and histone ELISAs to determine apoptosis. As shown in Fig. 1
, administration of Ad-TRAIL, in contrast to Ad-FasL, did not result in significant apoptosis in healthy livers.
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To investigate whether TRAIL-mediated apoptosis of hepatocytes in vivo needs triggering through virus infection, we treated mice with 5 x 107 pfu/g of Ad-lacZ 24 h before administration of 1 x 107 pfu/g of Ad-FasL or Ad-TRAIL. As shown in Fig. 1
pretreatment with 5 x 107 pfu/g Ad-lacZ does not enhance FasL (CD95L)-mediated apoptosis, but TRAIL-mediated apoptosis is strongly triggered through the viral infection. After application of 5 x 107 pfu/g Ad-LacZ, TRAIL-mediated apoptosis was at least at the level of FasL (CD95L)-mediated apoptosis.
TRAIL and TRAIL receptors are up-regulated in viral hepatitis and hepatitis-mediated acute liver failure
Protein expression of TRAIL and TRAIL receptors were assessed in normal liver tissue and tissues derived from patients who underwent orthotopic liver transplantation due to hepatitis C-mediated liver cirrhosis or hepatitis B-mediated acute liver failure. TRAIL expression was up-regulated in all cases of hepatitis B-mediated acute liver failure and in 4 of 5 cases of chronic hepatitis C-mediated liver cirrhosis (Fig. 2
). In contrast to chronic hepatitis C-infected livers, 4 of 5 livers with hepatitis B-mediated acute liver failure showed overexpression of TRAIL receptor DR 5.
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TRAIL and TRAIL receptor DR5 in adenovirus-mediated acute liver failure in mice
To further elucidate the role of TRAIL and TRAIL receptor DR5 in viral hepatitis, we next investigated protein expression in an animal model of adenovirus-mediated hepatitis. Application of 1 x 1010 pfu/g of Ad-LacZ in mice leads to acute liver failure. Mice were killed 40 h after viral infection and the livers were harvested for ß-Gal assays and Western blot experiments. A single treatment with high titer Ad-LacZ leads to up-regulation of TRAIL receptor DR5, but not of TRAIL, in the liver of balb/c and NMRI nude mice.
Because replication incompetent adenovirus mediates a very short stress response, which may not reflect the course of acute liver failure on viral infections in humans, the effects of repeated application of viral vectors were investigated. On day one 1 x 109 pfu/g, on day two 3 x 109 pfu/g, and on day five 5 x 109 pfu/g (total amount of 9x109 pfu/g) of Ad-lacZ were injected into the tail vein of mice. Repeated application of adenoviral vectors leads to strong up-regulation of TRAIL, in balb/c as well as in NMRI nude mice. To explore a possible contribution of Kupffer cells and NK cells on up-regulation of TRAIL during viral hepatitis, both kinds of cells were depleted in NMRI nude mice, which were subsequently treated with repeated application of adenovirus. Depletion of Kupffer cells by GdCl3 did not influence TRAIL expression, whereas depletion of NK cells by anti-asialoGM1 Ab even enhanced the TRAIL expression during adenoviral hepatitis. To exclude that TRAIL overexpression was the result of liver regeneration, 2/3 partial hepatectomy was performed in mice. In contrast to adenoviral infection, sham operation or 2/3 hepatectomy does not up-regulate TRAIL expression in the liver.
TRAIL receptor DR5 is transcriptionally up-regulated after viral infection
To asses whether the increase of TRAIL receptor DR 5 expression after viral infection was transcriptionally mediated, a reporter plasmid with a luciferase gene linked to the human TRAIL-DR5 promoter was transiently transfected in hepatoma cells. Two hepatoma cell lines with different p53 status were investigated (HepG2 (p53wt) and Huh7 (p53-220C mutation). The TRAIL-DR5 promoter was strongly activated after adenoviral infection in both cell lines.
CONCLUSION AND SIGNIFICANCE
The receptor/ligand pair Fas (CD95)/FasL (CD95L) has been noted to play an important role in the apoptosis of hepatocytes and in acute liver failure. In this paper, we describe a novel mechanism for virus-mediated acute liver failure involving the up-regulation of TRAIL receptor DR5 and TRAIL. We observed overexpression of TRAIL and TRAIL receptor DR5 in human hepatitis-mediated acute liver failure and a mouse model of adenoviral hepatitis. Thus, in both species the TRAIL receptor/ligand pair is up-regulated in virus-mediated acute liver failure. We show that TRAIL receptor DR5 is overexpressed in the early phase of an acute viral-mediated stress response whereas TRAIL is highly up-regulated in the liver after repeated application of adenoviral vectors. We could not observe TRAIL receptor DR5 up-regulation in the liver of patients with chronic hepatitis C or in mice after repeated administration of low doses of adenoviral vectors, which may be explained by the lower stress response in these livers. However, reporter gene experiments revealed that TRAIL receptor DR5 is transcriptionally up-regulated through adenoviral infection, which may provide one molecular explanation for virus-mediated TRAIL receptor DR5 overexpression.
In contrast to FasL (CD95L), which is known to induce a constitutive death signal in hepatocytes, we show that TRAIL-mediated apoptosis of murine hepatocytes in vivo needs triggering through viral infection. Consequently, we propose a model in which virus infection of the liver results in up-regulation of TRAIL-promoting receptors and overexpression of TRAIL, thereby activating the TRAIL-specific death pathway solely in infected hepatocytes.
Since TRAIL overexpression after viral infection is not dependent on lymphocytes, Kupffer cells, or NK cells, the TRAIL receptor/ligand system appears to be a paracrine mechanism of hepatocytes against viral infections. We propose that not only cellular transformation but also viral infection triggers sensitivity for TRAIL-mediated apoptosis as a component of an innate defense mechanism. The overexpression of TRAIL in hepatitis might be a defensive mechanism of the organism to eliminate infected cells and limit viral replication. Our results demonstrate that TRAIL-mediated hepatotoxicity has to be considered in tumor therapy of patients with viral hepatitis or other liver diseases that may activate TRAIL sensitivity in hepatocytes. However, these results also suggest that TRAIL might be a new attractive candidate for cytokine therapy of chronic viral hepatitis that can effectively eliminate infected hepatocytes and limit viral replication but may be harmless to normal uninfected hepatocytes in vivo.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0537fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0537fje 
2 Both authors contributed equally to this work.
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