Volume 119, Issue 2, Pages 446-460 (August 2000) Tumor necrosis factor α in the pathogenesis of human and murine fulminant hepatic failure  Konrad Streetz, Ludger Leifeld, Danja Grundmann, Jan Ramakers, Kolja Eckert, Ulrich Spengler, David Brenner, Michael Manns, Christian Trautwein  Gastroenterology  Volume 119, Issue 2, Pages 446-460 (August 2000) DOI: 10.1053/gast.2000.9364 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) TNF-α, (B) sTNF-R1, and (C) sTNF-R2 receptor levels are increased in serum from patients with FHF. Serum was obtained fom controls (C) and FHF patients. Specific time points are shown for patients with FHF: directly at admission (A), time point of decision when the patients recovered or either died or underwent transplantation (D), and 2 days before the time point of decision (2d-D). Subgroups consisted of patients who either recovered and those who died or underwent transplantation. *P < 0.05. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Higher expression of TNF receptors in the livers of patients with FHF. Liver sections of NL, CLD (photographs for CLD are not shown), or FHF were stained for the expression of (A, B, C, and D) TNF-R1 and (E, F, and G) TNF-R2. Representative liver sections of FHF (A), CLD (not shown), and NL (B) were stained for TNF-R1. (C) The average of TNF-R1–positive hepatocytes (#P < 0.0001, *P = 0.001) and (D) all TNF-R1–positive cells per field (#P = 0.03, *P = 0.0005). TNF-R2 expression in liver sections derived from FHF (E), CLD, and NL (F). (G) Statistical analysis of all liver sections for TNF-R2 (#P < 0.06, *P < 0.0001). Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Higher TNF-α expression on nonparenchymal liver cells correlates with an increase in apoptotic hepatocytes in livers from patients with FHF. Liver sections of NL, CLD (photographs not shown), or FHF were investigated for (A, B, and I) expression of TNF-α or (C, D, J, and K) presence of apoptotic cells by TUNEL assay. Staining for TNF-α expression was performed in liver sections of FHF (A), CLD (not shown), and NL (B). (I) Statistical analysis for TNF-α expression for the 3 groups (#P < 0.0001, *P < 0.0001). Apoptosis was detected by TUNEL assay in liver samples of FHF (C), CLD (not shown), or NL (D). (J) Statistical analysis for TUNEL-positive hepatocytes (#P = 0.003, *P = 0.001). (K) Correlation between TNF-α expression and TUNEL-positive cells (r = 0.63, P < 0.0001). Liver sections of FHF patients were double-stained for TNF-α expression (E) and the cell marker (macrophages) CD68 (F) to show that these macrophages are the main source of endogenous TNF-α production. Serial stainings showed that macrophages (H) located adjacent to TNF-R1–positive hepatocytes (G) expressed TNF-α. TNF-α expression was mainly evident periportal and in areas with high density of infiltrating cells. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Higher TNF-α expression on nonparenchymal liver cells correlates with an increase in apoptotic hepatocytes in livers from patients with FHF. Liver sections of NL, CLD (photographs not shown), or FHF were investigated for (A, B, and I) expression of TNF-α or (C, D, J, and K) presence of apoptotic cells by TUNEL assay. Staining for TNF-α expression was performed in liver sections of FHF (A), CLD (not shown), and NL (B). (I) Statistical analysis for TNF-α expression for the 3 groups (#P < 0.0001, *P < 0.0001). Apoptosis was detected by TUNEL assay in liver samples of FHF (C), CLD (not shown), or NL (D). (J) Statistical analysis for TUNEL-positive hepatocytes (#P = 0.003, *P = 0.001). (K) Correlation between TNF-α expression and TUNEL-positive cells (r = 0.63, P < 0.0001). Liver sections of FHF patients were double-stained for TNF-α expression (E) and the cell marker (macrophages) CD68 (F) to show that these macrophages are the main source of endogenous TNF-α production. Serial stainings showed that macrophages (H) located adjacent to TNF-R1–positive hepatocytes (G) expressed TNF-α. TNF-α expression was mainly evident periportal and in areas with high density of infiltrating cells. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 The FADDdn virus protects primary mouse hepatocytes from TNF-α– and Fas-induced apoptosis. (A) Primary mouse hepatocytes were isolated, plated, and infected with the Ad constructs as indicated. Twelve hours after infection, cells were stimulated for 18 hours with 50 ng/mL TNF-α. Morphologic changes such as membrane blebbing and cell shrinking indicate apoptotic cells. Ad-I-kB-AA and to a minor degree Ad-TRAF2dn sensitized hepatocytes against TNF-α, but no changes in cell morphology were evident in uninfected or Ad-β-gal– and Ad-FADDdn–infected cells. (B) To quantitate the amount of apoptotic cells, mouse hepatocytes were scraped 18 hours after stimulation with TNF-α and further analyzed for DNA fragmentation by histone-ELISA (Boehringer Mannheim). The DNA fragmentation rate of uninfected hepatocytes before treatment was set to 1. Changes compared with control cells are shown as fold activation. (C) Gel shift experiments were performed with nuclear extracts derived from primary hepatocytes and an oligonucleotide representing the NF-κB consensus site. The cells were either control treated or infected with Ad-β-gal, Ad-I-kB-AA, Ad-TRAF2dn, or Ad-FADDdn. Primary hepatocytes were stimulated for 30 minutes with 50 ng/mL TNF-α when indicated. The indicated bands represent NF-κB–specific complexes, as determined by supershift experiments (data not shown). (D) Primary mouse hepatocytes were infected with the Ad-β-gal or the Ad-FADDdn virus as indicated. Twelve hours after infection, cells were either treated with the buffer control (PBS), TNF-α (50 ng/mL), or Jo-2 antibody (0.1 μg/mL) and CHX (10 μg/mL) for 18 hours. Ad-β-gal–infected cells showed the typical signs of apoptosis, but hepatocytes pretreated with the FADDdn virus were protected from either TNF-α– or Jo-2–induced apoptosis. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 The FADDdn virus protects primary mouse hepatocytes from TNF-α– and Fas-induced apoptosis. (A) Primary mouse hepatocytes were isolated, plated, and infected with the Ad constructs as indicated. Twelve hours after infection, cells were stimulated for 18 hours with 50 ng/mL TNF-α. Morphologic changes such as membrane blebbing and cell shrinking indicate apoptotic cells. Ad-I-kB-AA and to a minor degree Ad-TRAF2dn sensitized hepatocytes against TNF-α, but no changes in cell morphology were evident in uninfected or Ad-β-gal– and Ad-FADDdn–infected cells. (B) To quantitate the amount of apoptotic cells, mouse hepatocytes were scraped 18 hours after stimulation with TNF-α and further analyzed for DNA fragmentation by histone-ELISA (Boehringer Mannheim). The DNA fragmentation rate of uninfected hepatocytes before treatment was set to 1. Changes compared with control cells are shown as fold activation. (C) Gel shift experiments were performed with nuclear extracts derived from primary hepatocytes and an oligonucleotide representing the NF-κB consensus site. The cells were either control treated or infected with Ad-β-gal, Ad-I-kB-AA, Ad-TRAF2dn, or Ad-FADDdn. Primary hepatocytes were stimulated for 30 minutes with 50 ng/mL TNF-α when indicated. The indicated bands represent NF-κB–specific complexes, as determined by supershift experiments (data not shown). (D) Primary mouse hepatocytes were infected with the Ad-β-gal or the Ad-FADDdn virus as indicated. Twelve hours after infection, cells were either treated with the buffer control (PBS), TNF-α (50 ng/mL), or Jo-2 antibody (0.1 μg/mL) and CHX (10 μg/mL) for 18 hours. Ad-β-gal–infected cells showed the typical signs of apoptosis, but hepatocytes pretreated with the FADDdn virus were protected from either TNF-α– or Jo-2–induced apoptosis. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 FADDdn blocks fulminant hepatic failure in vivo. BALB/c mice were either infected with Ad-β-gal or Ad-FADDdn. After 24 hours, mice were treated with 5 μg TNF-α and 15 mg gal. (A) Transaminases (ALT and aspartate aminotransferase) were determined before and at different time points after treatment. Each time point represents a group of 4 animals. (B) DNA fragmentation was determined in TNF-α/gal–injected animals as an indicator of apoptosis. Livers of the animals infected with Ad-β-gal or Ad-FADDdn virus and treated with TNF-α/gal were homogenized, and DNA fragmentation was determined by ELISA cell death detection system. The DNA fragmentation rate of β-gal–infected mice before treatment was set to 1. The other values were calculated accordingly and expressed as fold activation. (C) TUNEL staining of apoptotic cells: 5-μm cryosections were labeled with fluorescent nucleotides by terminal transferase (TUNEL technique). Apoptotic cells were visualized under a fluorescent microscope (Olympus AX 60; Hamburg, Germany, original magnification 400×). As a positive control, DNase-treated sections were used (data not shown). The β-gal-TNF-α/gal appears with a much darker background because of automatic exposure of the images to the microscope camera. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 FADDdn selectively blocks TNF-α–dependent apoptosis in vivo. (A) Cytosolic cytochrome c release. Cytosolic liver extracts were prepared from mice infected with the β-gal or the FADDdn virus and treated with either the buffer control or 5 μg TNF-α and 15 mg gal for 8 hours. Cytosolic extracts (75 μg) were used for Western blot analysis using anti–cytochrome c antibodies. As controls, extracts derived from untreated mice (lane 2) or rat heart cytochrome c (C; lane 1) were included. (B and C) Hepatic caspase activity. Supernatants of liver homogenates were used for measuring caspase-3 (B) and caspase-1 (C) activities. Animals were infected with Ad-β-gal or Ad-FADDdn virus and treated with TNF-α/gal for time points as indicated. Caspase activity as determined for the β-gal–infected animals was set to 1; other values are reported as fold activation. Gastroenterology 2000 119, 446-460DOI: (10.1053/gast.2000.9364) Copyright © 2000 American Gastroenterological Association Terms and Conditions