Tumor necrosis factor α, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver

GASTROENTEROLOGY2002;122:202-210

Tumor Necrosis Factor But Not Fas, Mediates Hepatocellular Apoptosis in the Murine Ischemic Liver H A N N E S A. RODIGER and PIERRE-ALAIN CLAVIEN Department of Surgery, Duke University Medical Center, Durham, North Carolina; and Division of Visceral Surgery & Transplantation, University Hospital Zurich, Zurich, Switzerland

Background&Aims: Apoptosis of hepatocytes is a central feature of ischemic injury in the liver. The aim of this study was to identify extracellular inducers of apoptosis in the murine ischemic liver. Methods: Involvement of tumor necrosis factor (TNF)-oL and Fas signaling was evaluated using various knockout mice (TNF-receptor 1 [ T N F - R 1 ] - / - , F a s [ I p r ] - / - , and Fas l i g a n d [ g l d ] - / - ) and wild-type mice pretreated with pentoxifylline, an inhibitor of TNF-oL synthesis. Results: Expression of TNF-oL was increased after ischemia and reperfusion in wild-type mice and TNF-Rl-deficient mice when compared with sham-operated animals. Pentoxifylline prevented up-regulation of TNF-oL expression. Inhibition of TNF-oL resulted in significant decrease of serum aspartate aminotransferase levels and prolonged animal survival. Markers of apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend labeling staining, cytochrome C release, and caspase 3 activity) were consistently decreased, and animal survival was prolonged after blocking TNF-oL. In contrast, inhibition of Fas signaling did not alter parameters of tissue injury or apoptosis, and animal survival remained unchanged. Conclusions: We identify TNF-~ as a crucial inducer of apoptotic cell death in the ischemic liver. A role for Fas could not be identified. These findings may lead to novel strategies to prevent ischemic injury of the liver.

lthough apoptosis (programmed cell death) is increasingly recognized as the key feature of cell death in the ischemic liver, 1 3 the mechanisms involved in the induction of apoptosis have remained elusive. For example, it is not known whether soluble cell death cyt0kines such as tumor necrosis factor (TNF)-o~4"5 or Fas ligand (FasL)6-8 are involved in the massive hepatocellular apoptosis occurring after reperfusion. TNF-o~ and FasL mediate apoptotic cell death through interaction with structurally related receptors belonging to the TNF/nerve growth factor super-family. 9 11 TNFreceptor 1 (TNF-R1) and Fas receptor are connected to a cytoplasmatic region called "death domain," which activates specific caspases with subsequent selective release of

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mitochondrial proteins. 12 These processes lead to morphologic changes of cellular structures, as well as to degradation of the chromosomal D N A with subsequent cell death. Several reports have incriminated TNF-ci in the pathogenesis of hepatic ischemia and reperfusion injury. For example, increased serum TNF-ot levels have been documented after reperfusion in a rat model of hepatic ischemia, and the levels correlated with the duration of ischemia. *~ Further studies have shown that TNF-ci mediates remote organ injury after prolonged ischemic injury to the liver. 14,15 In addition, the application of anti-TNF-o~ serum has been shown to reduce serum transaminase levels after hepatic ischemia. 13 Two distinct isoforms of the membrane receptors for TNF-o~ (TNF-R1 and TNF-R2) have been identified and molecularly cloned. Although most cell lines and primary tissues express both isoforms, 16 most of the biological activities of TNF-o~ are mediated through TNFR1.1v-*9 TNF-R2 is a poor inducer of apoptosis, 2° and binding affinities of soluble TNF-o~ are significantly higher to T N F - R 1 ) 8 First described in 1989, 21 the interaction between Fas receptor and FasL has become the best-characterized extracellular system triggering apoptosis, s,ll Accumulating data have incriminated this interaction in the pathogenesis of many gastrointestinal diseases including hepatitis, 2e,-~3 inflammatory bowel diseases, e4 and organ rejection. M,e5 In addition, recent data suggest that Fas receptor is involved in the pathogenesis of injury in the post-ischemic myocardium. 26 However, no data are cur-

Abbreviations used in this paper: AFU, arbitrary fluorescence units; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; FasL, Fas ligand; PCNA, proliferating cell nuclear antigen; TNF, tumor necrosisfactor; TNF-R1,TNF-receptor1; TUNEL,terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. © 2002 by tbe American GastroenterologicalAssociation 0016-5085/02/$35.00 doi:10.1053/gast.2002.30304

January 2002

rently available on the role of Fas receptor and FasL in the ischemic liver. Therefore, we designed a study to evaluate the 2 death-receptor systems T N F - i x / T N F - R 1 and Fas/FasL in the initiation of apoptotic cell death in the ischemic liver. Several strategies were used to inhibit these 2 signaling pathways, including genetically altered mice (TNF-R1-/-, F a s - / - , and F a s L - / - ) and pentoxifylline as p o t e n t inhibitor of TNF-ot p r o d u c t i o n s

Methods An in vivo mouse model of partial hepatic ischemia was used to evaluate TNF-cl and Fas signaling pathways as inducers of apoptosis. For each receptor/ligand system, 2 different ways of inhibition were used. TNF-o~ was evaluated using mice deficient in TNF-R1 and by treating wild-type mice with pentoxifylline. Pentoxifylline is a methylxanthine, which is a well-established inhibitor of TNF-c~ production in vivo.27 29 Fas signaling was evaluated using mice genetically deficient in either Fas(lpr) or FasL(gld).

Animals Pathogen-free male mice (C57BL6, TNF-R1 [ - / - ] , Fas [lpr, - / - ] , and FasL [gld, - / - ] ; Jackson Lab, Bar Harbor, ME) were used. Animals were fed on a laboratory diet with water and food ad libitum until use and were kept under controlled environmental conditions with a 12-hour light-dark cycle. All procedures were performed in accordance with Duke Institution Animal Care Committee guidelines.

Partial Hepatic Ischemia For morphologic and biochemical evaluation of tissue injury, we used a model of partial hepatic ischemia in mice with 75 minutes of ischemia and 3 hours of reperfusion as previously described) ° Briefly, after induction inhalation anesthesia with isoflurane, the abdominal cavity was opened through a midline incision, and the liver was freed from its ligaments. The portal triad, including portal vein, hepatic artery, and bile duct, was identified. Inflow occlusion to the median and left lobes was performed by placing a microvascular clamp (Aesculap, San Francisco, CA) on the respective branches, preserving adequate blood flow to the right and caudate lobes. Reperfusion was initiated by removing the clamp. After reperfusion, the liver was fixed in vivo by flushing the portal vein with paraformaldehyde (4% in phosphatebuffered saline).

Total Hepatic Ischemia Animal survival was determined using a model of total hepatic ischemia, as previously described) 1 Briefly, isoflurane inhalation anesthesia was induced, and the abdominal cavity was accessed through a midline incision. Inflow occlusion of the median and left lobes was performed by placing a micro vascular clamp as described for the model of partial hepatic

TNF-~-INDUCED APOPTOSIS IN THE ISCHEMIC LIVER

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ischemia. After the ischemic interval, the clamp was removed and the caudate and right lobes were resected, leaving only ischemic tissue in place. The animals were monitored for 7 days, and survival was calculated using the Kaplan-Meier method.

Serum Aspartate Aminotransferase Serum levels of aspartate aminotransferase (AST) served as sensitive a marker of hepatocyte injury. Blood was drawn from the caval vein after 3 hours of reperfusion in separate experiments. Serum was separated by centrifugation (10 minutes with 8000g at 4°C). AST levels were measured using the serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson Inc., Rochester, NY).

Terminal Deoxynucleotide Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling Staining Characteristic fragmentation of D N A strands is generally accepted as a specific down-stream event of apoptosis. For sensitive quantification of apoptotic hepatocytes, we used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method, which labels the characteristic D N A double-strand breaks. The assay was performed using a commercial kit (Roche Diagnostics, Mannheim, Germany), and the instructions from the manufacturer were strictly followed.

TNF-oL Levels in Liver Tissue Tissue levels of TNF-ot were quantified using an enzyme-linked immunosorbent assay (ELISA) kit. Tissue was perfused in situ through the portal vein with saline. The liver was then harvested and homogenized in extraction buffer (Tris 50 mmol/L [ph 7.2], NaCI 150 mmol/L, Triton-X 100 1%, and a protease inhibitor cocktail [Complete Mini; Roche, Basel, Switzerland]). The homogenate was shaken on ice for 90 minutes and then centrifuged at 3000g and 4°C for 15 minutes. TNF-ot was detected using a commercial ELISA kit (Quantikine M TNFix; R&D Systems, Abingdon, UK) according to the manufacturer's guidelines.

Cytochrome C Release The release of cytochrome C from the mitochondria into the cytoplasm was evaluated by Western blot analysis of the cytosolic fraction. Fresh tissue was homogenized in assay buffer (HEPES-KOH 20 mmol/L, pH 7.4, KC1 10 mmol/L, MgC12 1.5 mmol/L, EDTA 1 mmol/L, ethylene glycolbis[~aminoethyl ether]-N,N,N',N'-tetraacetic acid 1 mmol/L, dithiothreitol 1 mmol/L, phenylmethylsulfonyl fluoride 0.1 mmol/L, and sucrose 250 mmol/L). After centrifugation at 400g for 5 minutes and at 12,000g for l0 minutes, proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitro-cellulose membrane. The membrane was probed with a polyclonal anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immuno-

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Statistical Analysis

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Values are expressed as means + SD. Data was analyzed using SPSS software version 9.0.0 (SPSS Inc., Chicago, IL). Differences between groups were evaluated using the Student t test or analysis of variance (ANOVA) with Tukey's post hoc test. Animal survival was evaluated using the KaplanMeyer method and compared using the log rank test. Differences were considered statistically significant if P < 0.05.

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Do Tissue TNF-o~ Levels Increase in the Ischemic Liver?.

Figure 1. Tissue levels of TNF-(~ were significantly elevated after reperfusion in wild-type and T N F - R 1 - / - mice when compared with the controls (sham-operated animals). Levels in animals pretreated with pentoxifylline were significantly lower and comparable with levels in control animals (P < 0.001, ANOVA). *P < 0.001, Student t test when compared with either wild-type or T N F - R 1 - / - mice; n = 5 in each group.

precipitates were detected using a rabbit-anti-mouse antibody and enhanced chemiluminescence (Amersham, Piscataway,

NJ).

Production of TNF-oL was assessed in liver tissue using a specific ELISA kit (Quantikine; R & D Systems, Minneapolis, MN). As detailed in Figure 1, levels were low in sham-operated animals. After subjecting the animals to 75 minutes of ischemia and 3 hours of reperfusion, levels were significantly increased in wild-type mice and T N F - R l - d e f i c i e n t mice. However, the levels remained low after t r e a t m e n t with pentoxifylline before ischemia and reperfusion.

Caspase Assay Caspase-like activities were measured using an enzymatic, fluorometric assay. Samples were briefly sonicated (Sonitier 450; Branson, Danbury, CT) in assay buffer (50 mmol/L HEPES, p H 7.4, 100 mmol/L NaC1, 0.1% CHAPS [3-[(3cholamidopropyl)dimethylammonio]- l-propane-sul fonat el, 10 mmol/L dithiothreitol, 1 mmol/L EDTA, 10% glycerol). After centrifugation at 14,000g for 10 minutes, samples were incubated with a specific substrate (Ac-DEVD-AFC; Biomol, Plymouth Meeting, PA) for 3 hours. Fluorescence was measured (Fluostar; Biomedizintechnik, Offenburg, Germany), and readings were compared with values after incubation with a specific inhibitor (DEVD-CHO; Biomol) using an excitation filter for 400 nm and an emission filter for 505 nm.

Does TNF-~ Signaling Pathway Mediate Hepatocyte Injury in the Ischemic Liver? The impact of TNF-ot on hepatocyte injury was evaluated using T N F - R l - d e f i c i e n t mice ( T N F - R I - / - ) and p r e t r e a t m e n t of wild-type mice with pentoxifylline. Each animal was subjected to 75 minutes of hepatic ischemia. Serum levels of AST, an established marker of hepatocyte injury, 32 were measured after 3 hours of reperfusion. Both strategies resulted in dramatic decreases in serum AST levels (Figure 2). T h e lowest levels were observed in 6000 5000

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Proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) staining were used to quantify hepatic regeneration 2 days after resection of 30% of the liver mass. For this study, an ischemic period of 75 minutes would be associated with a high mortality rate in control animals. Therefore, we subjected the animals to only 50 minutes of ischemia, because this insult is not associated with any mortality in the control group. The rate of D N A synthesis correlates with the proliferation of the cells. Commercial kits were used to quantify PCNA expression (InnoGenex Inc., San Ramon, CA) and BrdU incorporation (Zymed Inc., San Francisco, CA) in liver sections. The numbers of PCNA-positive hepatocytes and hepatocytes with BrdU incorporation were determined in 15 high-power fields (400×). Data were expressed as percentage of positive hepatocytes per total number of hepatocytes.

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Figure 2. Serum AST levels after 75 minutes of ischemia and 3 hours of reperfusion were significantly lower in T N F - R 1 - / - mice and after pentoxifylline treatment, when compared with controls (sham-operated wild-type mice) (P < 0.001, ANOVA). Fas or FasL deletion had no effects on AST levels (P = 0.375, ANOVA). *P --< 0.05, Student t test when compared with controls, n - 5 in each group.

January 2002

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TNF-ec-INDUCEDAPOPTOSIS IN THE ISCHEMIC LIVER 205

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animals pretreated with pentoxifylline (718 + 58 U/L). TNF-Rl-deficient mice were also associated with low levels (1248 - 210 U/L). These figures were significantly lower than in controls (5271 -+- 278 U/L; P < 0.001, ANOVA). Does TNF-oL Induce H e p a t o c y t e Apoptosis in the Post-lschernic Liver?

TNF-(x induces apoptosis of hepatocyte through a caspase-dependent pathway in various types of injury. 33-35 Here, apoptosis was evaluated using several markers and mediators of the apoptotic pathway. The TUNEL method, which detects DNA strand breaks, was used to quantify the number of apoptotic hepatocytes. The release of cytochrome C from the mitochondria into the cytoplasm3<37 and the activation of the cystein pro-

Figure 4. TUNEL staining of liver tissue subjected to 75 minutes of ischemia and 3 hours of reperfusion. Massive apoptosis was observed in wild-type mice, whereas the absence of TNF-c~ or TNF-R1 or pretreatment with pentoxifylline were highly protective.

tease caspase 338 served as markers of the apoptotic cell death in liver tissue. Both strategies inhibiting TNF-oL signaling were associated with a dramatic reduction in TUNEL-positive hepatocytes after reperfusion (Figures 3 and 4): Although 82% - 7% of hepatocytes were apoptotic in wild-type mice after reperfusion, this figure was significantly lower in TNF-Rl-deficient mice (18% + 11%; P --< 0.001 compared with the controls, Student t test) and in wildtype mice pretreated with pentoxifylline (3% + 2%, P --< 0.001). The release of cytochrome C from the mitochondria into the cytoplasm and the assessment of caspase 3 activities were consistent with the results of the TUNEL assay. Massive release of cytochrome C was observed in control animals (wild-type mice subjected to 75 minutes of ischemia and 3 hours of reperfusion). In contrast, minimal release was found in TNFR 1 - / - mice and in mice pretreated with pentoxifylline (Figure 5). Tissue levels of caspase 3 activity were significantly lower after 3 hours of reperfusion in T N F - R l - d e f i cient mice (1.2 ± 0.6 arbitrary fluorescence units

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GASTROENTEROLOGY Vol. 122, No. 1

(AFU)/mg protein/min; P = 0.006, Student t test) and after pretreatment with pentoxifylline (2.3 + 0.9 AFU/mg protein/min; P = 0.001), when compared with the controls (8.1 --- 1.4 AFU/mg protein/min; wild-type mice subjected to ischemia and reperfusion) (Figure 6). Does Inhibition of TNF-oL Signaling Prevent Animal Death After Prolonged Periods of Total Hepatic Ischemia?

The clinical relevance of the previous biochemical and histologic fndings is best assessed by studies using animal survival as endpoint. We used our recently described murine model of total hepatic ischemia > to test the effects of TNF-ct inhibition on animal survival. As previously described, 3~ all animals died within 2 days of surgery after an ischemic insult of 75 minutes. Survival was significantly improved in TNF-Rl-deficient mice (mean 43.1 + 8.0 hours, P = 0.037, log rank) and in pentoxifylline-treated mice (116.7 + 22.8 hours; P = 0.006) subjected to the same ischemic insult (24.9 + 3.2 hours) (n = 7 in each group) (Figure 7). Is Animal Survival in the 3 Groups Influenced by Hepatic Regeneration?

Our model of total hepatic ischemia used for the survival experiments included a 30% partial hepatectomy. Experiments were performed to evaluate regeneration using the PCNA and BrdU assays in wild-type, T N F - R 1 - / - , and pentoxifylline-pretreated mice. Only a mild increase in PCNA and BrdU staining (--<0.5%) was observed in each group in absence of ischemic injury.

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In contrast, significant hepatocyte proliferation was detected in wild-type animals subjected to 50 minutes of ischemia. Inhibition of TNF signaling revealed reduced hepatocyte proliferation (Figure 8). Therefore, regeneration does not explain improved survival either in TNFR 1 - / - mice or in mice pretreated with pentoxifylline. Does Fas or FasL Deletion Prevent Hepatocyte Apoptosis or Animal Death After Ischemic Injury to the Liver?

The impact of Fas signaling on hepatocyte injury was evaluated in Fas- and FasL-deficient mice using a similar approach as for TNF-cl. Comparable AST levels were observed after reperfusion in Fas- and FasL-deficient and wild-type mice (P = 0.38, ANOVA; Figure 2). These findings were consistent with animal survival evaluated with the model of total hepatic ischemia. 3t Survival time was similar in Fas- and FasL-deficient animals and wild-type mice (Figure 9). The role of Fas signaling in mediating hepatocyte apoptosis after ischemia and reperfusion was determined by evaluating apoptotic markers in Fas- and FasL-deficient mice after 3 hours of reperfusion. The number of TUNEL-positive hepatocytes was high and similar in Fas (84% --+ 6%), FasL (77% ----- 6%), and wild-type mice ( 8 2 % + 7%) (P = 0.24, ANOVA; Figure 3). Caspase 3 activity was unchanged after Fas (10.9 -+- 0.3 AFU/mg protein/min) or FasL (8.4 + 0.6 AFU/mg/min) deletion, when compared with the controls (8.1 + 1.4 AFU/mg/ rain) (P = 0.12, ANOVA; Figure 6).

January 2 0 0 2

TNF..~-INDUCED APOPTOSIS IN THE ISCHEMIC LIVER

207

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The cytokines TNF-ot and FasL are 2 logical candidates initiating apoptosis in the ischemic liver. Colletti =~ 5 0 et al. 14 have previously shown that an ischemic insult in 0 the liver results in the rapid release of TNF-ot; the degree N 40 U of elevation was found to correlate with the severity of m P=O.061 D. 3 0 liver and remote organ injuries. 41 Although this landQ I "r mark study convincingly incriminated TNF-ot in the ~ 2o pathophysiology of ischemic injury in the liver, the mechanisms by which TNF-ot cause injury have reo o. 10 mained elusive. Fas and FasL are other good candidates in z view of recent studies in the myocardium of mice lacking o 0 functional Fas. 26 These animals displayed marked reducWild type TNF-R1 -/Pentoxifylline tion in cell death after ischemia and reperfusion when compared with wild-type mice. B The central role of TNF-ot signaling in inducing apoptosis in hepatocytes in this study is highlighted by W ® * several findings. The absence of TNFR-1 or the inhibi" 8 U tion of TNF-ot release by pentoxifylline dramatically o tl ! o. 6 prevented the release of cytochrome C, the activation -Iof caspase 3, and DNA fragmentation. Furthermore, the • 4 decrease in these markers correlated with lower AST levels and improved animal survival, pointing out to o o. 2 apoptosis as the predominant mechanism of cell death in the ischemic liver. This study, as others using antim 0 apoptotic strategies such as inhibitors of calpain 3° or Wild type TNF-R1 -/Pentoxifylline caspases 3 or Bcl-2 overexpression, 42 shows that apoptosis, Figure 8. Effects of 30% hepatectomy and 50-minute ischemia on not necrosis, is the key mechanism of injury in the hepatic regeneration, Inhibition of TNF signaling significantly reduced ischemic liver. For example, high levels of Bcl-2 comhepatic regeneration as assessed by (A) PCNA staining and (B) BrdU pletely blocked apoptosis after normothermic ischemia incorporation. *P < 0.001 compared with each of the 2 other groups, ANOVA/Tukey's. and significantly reduced liver injury and increased animal survival. 42 These data are in contrast with the recent

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Discussion

This study shows a pivotal role for TNF-cl in initiating apoprosis in the murine ischemic liver. Both models using TNF-Rl-deficient mice and wild-type mice receiving pentoxifylline disclosed resistance to apoptosis with increased survival after prolonged periods of ischemia. In contrast, this study failed to identify a role for the Fas/FasL pathway in the mechanisms of injury in the ischemic liver. Studies from our group 3° and others 3 have recently pointed out hepatocyte apoptosis as a critical mechanism of reperfusion injury in the ischemic liver. The cellular pathways of hepatocyte apoptosis during reperfusion have been well described with an early release of cytochrome C from the mitochondria into the cytoplasm followed by activation of the caspase cascade including caspase 3, and finally fragmentation of DNA with subsequent cell death.39, 4o However, it has remained unclear whether extracellular mediators of injury are involved.

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75 minutes of total hepatic ischemia compared with the controls (wild-type). P > 0.5 if F a s - / - or F a s L - / - is compared with the control, log rank. n = 7 in each group.

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RODIGERAND CLAVIEN

report by Gujral et al., 43 which suggested that necrosis was the predominant form of injury. The use of 2 independent approaches (i.e., knockout mice and pentoxifylline treatment) to evaluate TNF signaling provides convincing evidence for a critical contribution of this signaling system. Although TNF-R 1 lacking mice had normal levels of TNF-o~, signaling is prevented by lack of the major TNF-ot receptor. Pretreatment with pentoxifylline was associated with complete absence of TNF-oL elevation. Of note, the use of pentoxifylline in wild-type mice resulted in deeper prevention of injury than in TNF-Rl-deficient mice, including improved animal survival. This observation might be related to 4 different factors. First, TNF-o~ acts not exclusively on TNF-R1 but also, to a lesser part, on related receptors of the TNF superfamily including TNF-R2. 44 Second, TNF-Rl-deficient mice might develop alternative apoptotic pathways in compensation of the deficient TNF-R1. For example, we recently noted that chronic manipulation of distinct mediators of the apoptotic pathway (e.g., by over-expressing the anti-apoptotic protein Bd-2) induces a compensatory up-regulation of cytochrome C release and caspase 3 activity. 42 Third, pentoxifylline may prevent reperfusion injury by additional mechanisms unrelated to TNF-oL inhibition such as improved tissue perfusion, 2v inhibition of platelet adhesion, 27 and free radical scavenging properties. 45,46 Such additional mechanisms might be important for future clinical application of pentoxifylline in patients undergoing major liver surgery. Fourth, impaired hepatic regeneration in TNF-R1 - / - mice, as recently reported by Yamada et al., 4v could have a negative effect on animal survival. However, our data indicate that regeneration after resection and ischemia were not significantly different after TNF-R1 deletion or pentoxifylline pretreatment. The impaired regeneration observed in TNFR 1 - / - mice and in mice pretreated with pentoxifylline further supports the role of TNF signaling in mediating hepatocyte injury. Although we show that the engagement of TNF-o~ and TNF-R1 is tightly bound to the apoptotic cascade in hepatocytes after ischemia, the link between this membranous event and the release of cytochrome C from the mitochondria was not investigated in this study. Of interest, studies in various cell systems (including hepatocytes) revealed that TNF-ot alone is not a sufficient trigger for the cell to die. 48,49 It has been suggested that TNF-o~ is only cytotoxic in "pre"-sensitized cells. Accumulating evidence indicates that oxidative stress is necessary for some TNF-ot toxicity. 5° For example, the antioxidant n-acetylcysteine reduced TNF-ot toxicity in

GASTROENTEROLOGY Vol. 122, No. 1

rats, 51 and Xu et al. 48 found that depletion of the natural antioxidant glutathione is associated with increased mortality in a murine model of TNF-o~ cytotoxicity. These findings together with our data lead to the hypothesis that oxidative stress may sensitize the liver to TNF-oL, which in turn induces massive apoptosis. By the same token, oxidative stress might not be directly cytotoxic in the ischemic liver, but rather a "facilitator" of TNF-oLmediated cell death. This might represent the focus of further key studies in the field of ischemic injury. This study suggests that the Fas pathway is not involved in the pathogenesis of ischemic injury in the murine liver. Despite similarities between some cellular events after TNF-ot- and Fas-induced apoptosis in other models (e.g., release of cytochrome C and activation of caspase 3), our data indicate that the effects of TNF-~x in mediating injury are independent of Fas. This observation is in keeping with the recent report by Nagaki et al., 52 in which TNF-oL stimulation of the nuclear transcription factor KB in hepatocytes was independent of Fas signaling. In conclusion, TNF-c~ induces hepatocyte apoptosis upon reperfusion of the ischemic liver, an event independent of Fas signaling. This newly characterized role for TNF-ot may point towards novel protective strategies for patients undergoing liver surgery and transplantation, or after trauma. This may include the application of TNF-oL antibodies, soluble TNF receptor constructs, 53,54 and molecules inhibiting TNF-oL production or release such as pentoxifylline2v or thalidomide. 55

References 1. Kohli V, Selzner M, Madden J, Bentley R, Clavien P. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemiareperfusion injury in the rat liver. Transplantation 1999;67: 1099-1105. 2. Gao W, Bentley RC, Madden JF, Clavien PA. Apoptosis of sinusoidal endothelial cells is a critical mechanism of prevention injury in rat liver transplantation. Hepatology 1 9 9 8 ; 2 7 : 1 6 5 2 1660. 3. Cursio R, Guggenheim J, Ricci J, Crenesse D, Rostagno P, Maulon L, Saint-Paul M, Ferrua B, Auberger AP. A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis. FASEB J 1999;13:253-261. 4. Bradham C, Plumpe J, Manns M, Brenner D, Trautwein C. Mechanisms of hepatic toxicity. Am J Physiol 1998;275:G387-392. 5. Wallach D, Kovalenko A, Varfolomeev E, Boldin M. Death-inducing functions of ligands of the tumor necrosis factor family: a Sanhedrin verdict. Curr Opin Immunol 1 9 9 8 ; 1 0 : 2 7 9 - 2 8 8 . 6. Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itho N, Suda T, Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature 1 9 9 3 ; 3 6 4 : 8 0 6 - 8 0 9 . 7. Nagata S, Goldstein P. The Fas death factor. Science 1995;267: 1449 -1456. 8. Krammer P. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv Immunol 1999;71:163-210. 9. Smith C, Farrah T, Goodwin R. The TNF receptor superfamily of

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receptor of tumor necrosis factor to prevent acute and chronic experimental liver injury. Yale J Biol Med 1 9 9 7 ; 7 0 : 3 9 1 - 4 0 2 . 55. Lopez-Talavera J, Cadelina G, Olchowski J, Merrill W, Groszmann R. Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats. Hepatology 1996;23: 1616-1621. Received March 13, 2001. Accepted September 20, 2001. Address requests for reprints to: Pierre-A. Clavien, M.D., Ph.D., FACS, Professor of Surgery, Division of Visceral and Transplantation Surgery, University Hospital Ziirich, R~imistrasse 100, 8 0 9 1 Ziirich, Switzerland. e-mail: [email protected]; fax: 41 1 255 44 49. This study was supported by a grant from the National Institutes of Health (DK54048-OIA1) and from the Swiss National Science Foundation (SNF3200-061411) (to P.-A.C.). Dr. RiJdiger is supported by personal grants from the Swiss National Science Foundation (grant 81ZH-059608) and the Roche Research Foundation, Switzerland, and the Olga-Mayenflsch Foundation, Switzerland. Parts of this study were presented at the 51st Annual Meeting of the American Association for the study of Liver Diseases, October 27-31, 2000, Dallas, Texas. The authors thank Horst Bliithmann, Ph.D., from Roche Genetics, Basel, Switzerland, for providing us with knock-out animals.