Alcoholic fatty liver differentially induces a neutrophil-chemokine and hepatic necrosis after ischemia-reperfusion in rat

Alcoholic Fatty Liver Differentially Induces a NeutrophilChemokine and Hepatic Necrosis After Ischemia-Reperfusion in Rat SHINWA YAMADA,1 TAKESHI IIDA,2 TAKAHIRO TABATA,2 MINORU NOMOTO,3 HIROFUMI KISHIKAWA,2 KIMITOSHI KOHNO,3 AND SUMIYA ETO2

Primary graft nonfunction of steatotic liver allograft is one of the factors causing shortage of donor livers. Ischemia/ reperfusion (I/R) injury is an important contributory factor to primary graft nonfunction. In this study, we investigated the complex chain of events from transcription factor activation to necrosis through cytokine induction and apoptosis in steatotic rat liver after warm I/R. Rats with alcoholic or nonalcoholic fatty liver were subjected to hepatic warm I/R and compared with control rats. Rats fed an ethanol diet for 6 to 8 weeks developed severe hepatic necrosis accompanied by increased neutrophil recruitment after I/R, compared with rats with nonalcoholic fatty liver or control. Hepatic apoptosis as assessed by DNA fragmentation at 4 hours after I/R, however, increased to a similar degree in each of the 2 fatty liver models compared with the control. Alcoholic fatty liver exposed to I/R showed a rapid increase in nuclear factor-␬B (NF-␬B) binding activity at 1 hour after I/R, which preceded an increased expression of tumor necrosis factor ␣ (TNF-␣) and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In contrast, nonalcoholic fatty liver did not show such potentiation of either NF-␬B activation or cytokine induction after I/R. Our results have indicated that alcoholic fatty liver may differentially induce CINC-1 production and hepatic necrosis after I/R. Furthermore, our results suggest that apoptosis per se does not always lead to necrosis in the liver following I/R. (HEPATOLOGY 2000;32:278-288.) Primary graft nonfunction of steatotic liver allograft is one of the hurdles causing a shortage of donor livers.1-3 Ischemia/ reperfusion (I/R) injury, one of the important causes of priAbbreviations: I/R, ischemia/reperfusion; CINC-1, cytokine-induced neutrophil chemoattractant-1; NF-␬B, nuclear factor-␬B; AP-1, activator protein-1; ALT, alanine transaminase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; cDNA, complementary DNA; RT-PCR, reverse transcription-polymerase chain reaction; mRNA, messenger RNA; TNF-␣, tumor necrosis factor ␣; IL-1␣, interleukin 1␣. From the 1Department of Clinical Pathophysiology, 2First Department of Internal Medicine, 3Department of Molecular Biology, University of Occupational Environmental Health, Fukuoka, Japan. Received February 9, 2000; accepted May 31, 2000. Supported by Grant 10670523 for scientific research from the Ministry of Education, Science, and Culture of Japan. Address reprint requests to: Shinwa Yamada, M.D., Department of Clinical Pathophysiology, School of Health Sciences, University of Occupational Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu, Fukuoka 807-8555, Japan. E-mail: [email protected]; fax: (81) 93-691-9334. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3202-0016$3.00/0 doi:10.1053/jhep.2000.9604

mary graft nonfunction, is thought to play a significant role in the initiation of acute liver injury culminating in organ failure. Although the exact mechanisms of I/R injury remain elusive, a number of studies have suggested that activated Kupffer cells4,5 and neutrophils5-9 are critical for the induction of inflammation as well as sinusoidal endothelial cell death4,10,11 in I/R injury models. We paid close attention on activation and recruitment of neutrophils as a central event in the set of phenomena after I/R especially in alcoholic fatty liver, because a significant role of neutrophil was implied not only in posttransplantation liver injury but also in alcohol-induced liver injury. We and others have shown the involvement of neutrophil-chemokines in hepatic injury after transplantation or I/R in normal rodents.12-16 In our studies using ethanol-fed rats, we have shown that hepatic macrophages are activated even in fatty livers17 and that cytokine-induced neutrophil chemoattractant-1 (CINC1), the most potent neutrophil-chemokine in rats, might contribute to the development of hepatic necrosis by enhancing neutrophil recruitment.18 Transcription factors such as nuclear factor ␬B (NF-␬B) and activator protein-1 (AP-1) are involved in acute response of I/R liver injury.19-21 In this regard, CINC-1 gene is known to contain an NF-␬B– binding sequence,22 and indeed, NF-␬B activation leads to a transcriptional activation of the CINC-1 gene23 as well as genes encoding other inflammatory cytokines. The production of reactive oxygen species, which is responsible for NF-␬B activation, is thought to be potentiated in steatotic livers.24-26 Apoptosis is increasingly recognized as a distinct form of cell death even in a variety of pathologic conditions, including alcohol-induced liver disease as well as I/R injury.27,28 Reactive oxygen species were reported to be involved as major mediators of apoptosis in the above pathologic conditions. In addition, several lines of evidence suggest that apoptosis positively contributes to triggering inflammation and necrosis.11,29 Before identifying a key mechanism of potentiation of hepatic necrosis after I/R in fatty liver, it is worthwhile to outline a set of events leading to inflammation and necrosis, i.e., transcription factors, cytokines or chemokines, leukocytes, and apoptosis. The purpose of this study was to build a framework on which we attempt to find a breakthrough leading to a successful transplantation of steatotic livers. The present study was undertaken to determine whether CINC-1 induction after I/R is potentiated in steatotic liver, whether the transcription factor activation is associated with the subsequent hepatic injury, and whether apoptosis is associated with hepatic necrosis after I/R in rats. To identify alcohol-selective

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TABLE 1. Triglyceride and Cholesterol Contents (␮g/mg protein) in the Liver, and Percents of the Liver Weight Over Body Weight in Rats Fed an Ethanol Diet, a Cholesterol-Containing Control Diet, or a Dextrose-Control Diet

Alc Chol Dex

TG

Cholesterol

% LW/BW

355 ⫾ 68 429 ⫾ 82 83.8 ⫾ 14†

38.0 ⫾ 6.1 124 ⫾ 18* 17.3 ⫾ 3.1

4.10 ⫾ 0.15 4.27 ⫾ 0.16 3.52 ⫾ 0.05†

NOTE. Results are expressed as means ⫾ SEM of 5 nonfasted rats for each group. Hepatic triglyceride contents and percents of the liver weight were significantly higher in both fatty liver models (Alc and Chol) when compared with the control (Dex). Abbreviations: TG, triglyceride; LW/BW, liver weight over body weight; Alc, ethanol diet; Chol, cholesterol-containing diet; Dex, dextrose-control diet. *P ⬍ .01 vs. Alc or Dex by one-way ANOVA (Bonferroni). †P ⬍ .01 vs. Alc or Chol by one-way ANOVA (Bonferroni).

phenomena, we induced nonalcoholic fatty liver by feeding animals a cholesterol-containing diet in addition to the conventional dextrose control. This study may have general implications for the pathophysiology of I/R injury in other organs. MATERIALS AND METHODS Animals. Male Sprague-Dawley rats were obtained from a commercial supplier (Kyudo Inc., Kumamoto, Japan). All animals received humane care in compliance with the institution’s guidelines and criteria for humane care, as outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.30 These rats (weight-matched; 130-150 g) were fed an isocaloric liquid diet (Oriental Yeast Ind., Tokyo, Japan) containing 36% of the total calories as ethanol or dextrose with or without cholesterol at 1 mg/mL of the diet for 6 to 8 weeks according to the method of Lieber and DeCarli.31 Histologically, cholesterol-induced steatosis exhibited a mainly microvesicular appearance, whereas the ethanolinduced one exhibited both microvesicular and macrovesicular fatty liver as previously reported.24 To achieve a similar degree of fasting, the day before euthanasia, rats were provided with only 50% of the daily food intake at 11:00 AM and one sixth at 6:00 PM. The liquid diet was substituted with water at 9:00 PM. Hepatic Ischemia and Reperfusion. Under pentobarbital anesthesia, rats were heparinized (50 U/ kg body weight) via the penile vein. A midline laparotomy was performed and an atraumatic clamp was used to interrupt the arterial and portal venous blood flow to the median and left lobes of the liver. During ischemia for 30 minutes, the animals remained under anesthesia with a wet cheesecloth covering the opened abdomen under a heating lamp. After removing the clamp and confirming the restoration to normal color of the ischemic

279

lobes within 3 to 4 minutes, the abdomen was closed in 2 layers with silk. Under pentobarbital anesthesia, rats were sacrificed at 1, 4, and 24 hours after reperfusion, and a 2-mL blood sample was withdrawn from the inferior vena cava. The liver lobes exposed to I/R were carefully dissected and removed. Fifty and 100 mg of the liver sections were immediately frozen in liquid nitrogen. Sham-Operated Control. Sham-operated control rats underwent identical preparations, except that the atraumatic clamp was not applied. The abdominal wall was then closed with silk. Measurement of Hepatic Triglyceride and Cholesterol Contents. Triglyceride and cholesterol contents in the liver were measured using the method of Van Handel and Zilversmits32 with modifications as described previously,24 and the method of Rudel and Morris,33 respectively (5 nonfasted rats in each group). Measurement of Serum Transaminase Activity. Serum alanine transaminase (ALT) activity was measured using a standard clinical automatic analyzer (Hitachi 7170, Tokyo, Japan). Histologic Examination of the Liver. The liver was fixed with 10% (vol/vol) neutral-buffered formalin, processed, and embedded in paraffin. Tissue sections (5-␮m thick) were stained with hematoxylin-eosin for histologic evaluation by light microscopy. Four hours after reperfusion, the extent of liver necrosis was semiquantitatively assessed according to the area of the coagulative necrosis in the hepatic lobules as follows: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering less than 5%; grade 2, 5% to 20%; grade 3, more than 20%. Slides were evaluated by one investigator blinded to the treatments. Another section from the liver was stained with a chloroacetate esterase staining kit (Muto Pure Chemical Co., Tokyo, Japan) to determine the number of neutrophils that had infiltrated into the hepatic parenchyma. Neutrophil count was measured in 5 high-power fields in areas where overt coagulative necrotic areas were not included and expressed per square millimeter. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling Method. To detect apoptotic cells, another 5-␮m–thick section

from the liver was collected on a poly-L-lysine-coated glass slide, and the nuclear DNA fragmentation of apoptotic cells was labeled in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method34 as follows using an ApopTag Peroxidase In Situ Apoptosis Detection Kit (Intergen Co., Purchase, NY). Briefly, the section was deparaffinized and treated with 20 ␮g/mL proteinase K (Boehringer Mannheim, Mannheim, Germany) for 15 minutes. After rinsing with distilled water, the section was treated with 3% hydrogen peroxide in phosphate-buffered saline for 5 minutes. The section then was washed with distilled water and incubated in the provided equilibration buffer for 10 seconds. The section was incubated with terminal deoxynucleotidyl transferase in the provided reaction buffer in a humidifier chamber at 37°C for 60 minutes with the incorporated nucleotides forming an oligomer labeled with digoxigenin. The section was incubated with anti-digoxigenin peroxidase conjugate and then with diaminobenzidine solution according to the instructions provided by the manufacturer. After counterstaining with 0.5 % (wt/vol) methyl green (Chroma

TABLE 2. Serum ALT Level (IU/L) at 1, 4, and 24 Hours After Warm I/R in Rats Fed an Ethanol Diet, a Cholesterol-Containing Control Diet, or a Dextrose-Control Diet 1 Hr

Alc-I/R Alc-sham Chol-I/R Chol-sham Dex-I/R Dex-sham

2,377 ⫾ 602 103 ⫾ 48 1,465 ⫾ 366 69 ⫾ 10 1,304 ⫾ 389 71 ⫾ 15

4 Hr

(7) (3) (6) (3) (6) (3)

15,960 ⫾ 4,250* 121 ⫾ 24 4,256 ⫾ 993 54 ⫾ 14 3,397 ⫾ 799 100 ⫾ 30

24 Hr

(6) (3) (6) (3) (6) (3)

1,313 ⫾ 213† ND 476 ⫾ 244 ND 368 ⫾ 97 ND

NOTE. Results are expressed as means ⫾ SEM (number of rats). The mean ⫾ SEM of serum ALT activity of 5 normal male rats is 38 ⫾ 6 IU/L. Abbreviations: Alc, ethanol diet; Chol, cholesterol-containing diet; Dex, dextrose-control diet; ND, not done. *P ⬍ .02 vs. Chol or Dex by one-way ANOVA (Bonferroni) after logarithmic transformation of individual measurements. †P ⬍ .01 vs. Chol or Dex by one-way ANOVA (Bonferroni) after logarithmic transformation of individual measurements.

(7) (6) (6)

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TABLE 3. Extent of Hepatic Necrosis at 4 Hours After Warm I/R in Rats Fed an Ethanol Diet, a Cholesterol-Containing Control Diet, or a Dextrose-Control Diet

Alc-I/R* Chol-I/R Dex-I/R

Grade 0

Grade 1

Grade 2

Grade 3

Total

0 1 2

0 4 3

1 1 1

5 0 0

6 6 6

NOTE. Data are number of rats. The extent of liver necrosis was semiquantitatively assessed according to the area of the coagulative necrosis in the hepatic lobules as follows: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering less than 5%; grade 2, 5%-20%; grade 3, more than 20%. All livers of the sham-operated rats showed grade 0 (3 rats for each group). Abbreviations: Alc, ethanol diet; Chol, cholesterol-containing diet; Dex, dextrose-control diet. *P ⬍ .05 vs. Chol-I/R or Dex-I/R, by Mann-Whitney’s U test.

scribed above, and for supershift experiments with an antibody, 0.5 or 1 ␮L of anti-p50, p65 (a generous gift from Dr. Okamoto, Department of Molecular Genetics, Nagoya City University Medical School), c-fos or c-jun (Santa Cruz Biotech Inc., Santa Cruz, CA) was added to the nuclear extract, followed by incubation for 30 minutes at 4°C before initiation of the binding reaction with 32P-labeled oligonucleotide. Electrophoretic mobility shift assay (EMSA) and supershift reaction mixtures were then resolved by electrophoresis on native 4% polyacrylamide gels (39:1) in 0.5⫻ TBE buffer. Analysis of Messenger RNA by Reverse Transcription-Polymerase Chain Reaction or Northern Blotting. Total RNA was isolated from 100 mg of

the liver section frozen in liquid nitrogen by the method of Chomczynski and Sacchi38 using Trizol (GIBCO, Grand Island, NY) according to the manufacturer’s protocol. Two micrograms of total liver RNA was reverse transcribed to complementary DNA (cDNA) using Ready-To-Go T-Primed First-Strand Kit (Amersham Pharmacia Biotech Inc.) according to the protocol recommended by the

Gesellshaft, Koengen, Germany), the tissue section was viewed by light microscopy. Determination of Hepatic Histone-Associated Oligonucleosomal DNA Level. Histone-associated oligonucleosomal DNA was quantitated

using Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, 50 mg of the liver section frozen in liquid nitrogen was homogenized with 1 mL of the provided lysis buffer. The homogenate was centrifuged at 15,000g for 10 minutes and diluted supernatant was used for ELISA. The results were expressed as the percentage of histone-associated oligonucleosomal DNA over an average value obtained from 5 normal rat livers. Electrophoretic Analysis for DNA Fragmentation. Electrophoretic detection of DNA cleavage products was performed by the method of Roesl35 using an apoptotic laddering kit (Trevigen Inc., Gaithersburg, MD) according to the protocol recommended by the manufacturer and adapted for tissues. Briefly, 50 mg of the liver section frozen in liquid nitrogen was homogenized with 1 mL of a lysis buffer (10 mmol/L Tris-HCl [pH 7.5], 10 mmol/L ethylenediaminetetraacetic acid, 0.5% Triton X-100). One microgram of cellular DNA isolated from the liver homogenate was radiolabeled with the Klenow fragment of DNA polymerase I and 32P-␣-CTP and electrophoresed on a 1.5 % agarose gel. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays.

Nuclear extracts were prepared as described by Gorski et al.,36 after the isolation of nuclei from the rat livers. Nuclear extracts were quick-frozen and stored in aliquots at ⫺80°C until use. The oligonucleotides of the following sequences were used (factor binding sites are underlined): CINC–NF- ␬B: 5⬘-GCTCC GGGAATTTCC CTG GCC-3⬘, AP-1; 5⬘-CGCTTGA TGAGTCA GCCGGAA-3⬘. The above oligonucleotides and their complementary oligonucleotides were synthesized according to the sequence reported previously,22 then purified on 15% denaturing polyacrylamide gels. The singlestranded oligonucleotides were labeled at the 5⬘ end with [␥32P] adenosine triphosphate and T4 polynucleotide kinase (Takara, Shiga, Japan) and then annealed by heating and slow cooling. The double-stranded oligonucleotide was recovered from a 15% denaturing polyacrylamide gel. For the gel shift assay, 4 ␮g protein of the nuclear extract was mixed with 32P-labeled oligonucleotides and 1 ␮g of poly (dIdC) (Amersham Pharmacia Biotech Inc.) in 10 mmol/L Tris-HCl (pH 7.8) containing 50 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiotreitol, and 5% glycerol in a final volume of 25 ␮L as described previously.37 The binding reactions detecting CINC–NF- ␬B and AP-1 activities contained 2 mmol/L and 4 mmol/L MgCl2, respectively. The binding mixtures were allowed to stand for 30 minutes at room temperature and then analyzed on a native 4% polyacrylamide gel in 0.5⫻ TBE buffer. For competition experiments, a 200-fold excess amount of the indicated unlabeled oligonucleotide was added to the binding mixtures de-

FIG. 1. Effect of alcoholic or nonalcoholic fatty liver on neutrophil recruitment into the liver at 4 hours after I/R. (A) Number of infiltrating neutrophils at 4 hours after I/R. Data are expressed as means ⫾ SEM of 6 rats. *P ⬍ .05 versus cholesterol, and P ⬍ .01 versus dextrose, by one-way ANOVA (Bonferroni). Alc, ethanol-fed; Chol, cholesterol-fed; and Dex, dextrose-fed control. Number of the neutrophils at 4 hours after the sham operation was 34.7 ⫾ 14.7 (Alc), 44.4 ⫾ 8.88 (Chol) and 31.3 ⫾ 5.25 (Dex) (n ⫽ 3, each group). (B) A high-power photomicrograph showing numerous infiltrating neutrophils in the liver of a representative ethanol-fed rat at 4 hours after ischemia/reperfusion (chloroesterase staining specific for neutrophils; original magnification ⫻400).

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FIG. 2. A representative TUNEL-staining of the liver at 4 hours after I/R in a rat with alcoholic fatty liver. (A) In a few layers of hepatocytes surrounding coagulative necrotic areas were shown to be TUNEL-positive with dense condensation of nuclear chromatin at 4 hours after reperfusion (original magnification ⫻100). (B) TUNEL-positive hepatocytes were also scattered in the hepatic lobules (original magnification ⫻400).

manufacturer. An aliquot of the cDNA product from 400 ng of RNA was amplified with 1.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania), 1.5 mmol/L MgCl2, and 5 ␮mol/L of primer. After 5 minutes of initial melting at 94°C, the mixture was amplified for 25 and 30 cycles using a 3-step cycle process that began with melting at 94°C for 40 seconds, annealing at 60°C for 40 seconds, followed by extension at 72°C for 40 seconds. Five ␮L of each reverse-transcription polymerase chain reaction (RT-PCR) was electrophoresed on a 2% agarose gel and stained with ethidium bromide. Quantitation was performed by comparing the fluorescence intensities with those of rat ribosomal protein S6 transcript, and the data were expressed relative to the amount of S6 messenger RNA (mRNA) present in each specimen. PCR primers designed from cDNA sequences for CINC-1, rat ribosomal protein S6, rat tumor necrosis factor ␣ (TNF-␣) and rat interleukin 1␣ (IL-1␣) as follows: CINC-1,

sense 5⬘-GCTCGCTTCTCTGTGCAGCG-3⬘, antisense 5⬘-GCTTCAGGGTCAAGGCAAGCC-3⬘; ribosomal protein S6, sense 5⬘-GTCCGGATCAGCGGTGGG-3⬘, antisense 5⬘-CGT TTGTGTTGCAGGACACGG-3⬘; TNF-␣, sense 5⬘-GCCTCCAGAACTCCAGGCG-3⬘, antisense 5⬘-GGCACGCTGGCTCAGCC-3⬘; IL-1␣, sense 5⬘-CCTTTACTGAAGATG ACCTGGAGG-3⬘, antisense 5⬘-GTCTCCTCCCGATGAGTAGGC-3⬘. Twenty micrograms of total RNA was electrophoresed on 1% agarose-formaldehyde denaturing gels, and transferred to a Hybond N⫹ membrane (Amersham Pharmacia Biotech Inc.). The membrane was hybridized with 32Plabeled cDNA probes of CINC-1 containing the full length of its mRNA sequence (a generous gift from Dr. Konishi, Department of Biochemistry, Toyama Medical and Pharmaceutical University). The presence of a similar amount of total RNA was ensured by comparison of ribosomal RNA stained with ethidium bromide.

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TABLE 4. Hepatic Histone-Associated Oligonucleosomal DNA Levels at 1, 4, and 24 Hours After Warm I/R in Rats Fed an Ethanol Diet, a Cholesterol-Containing Control Diet, or a Dextrose-Control Diet 1 Hr

Alc-I/R Alc-sham Chol-I/R Chol-sham Dex-I/R Dex-sham

198 ⫾ 49.1 ND 134 ⫾ 5.48 ND 169 ⫾ 38.7 ND

4 Hr

(4) (3) (3)

939 ⫾ 153* 73.2 ⫾ 11.2 757 ⫾ 179‡ 89.1 ⫾ 24.5 309 ⫾ 34.0 103 ⫾ 22.4

24 Hr

(6) (3) (6) (3) (6) (3)

670 ⫾ 105† ND 104 ⫾ 22.7 ND 68.5 ⫾ 19.7 ND

(4) (4) (4)

NOTE. The results were expressed as the percentage of histone-associated oligonucleosomal DNA level over an average value obtained from 5 normal rat livers. Results are expressed as means ⫾ SEM (number of rats). Abbreviations: Alc, ethanol diet; Chol, cholesterol-containing diet; Dex, dextrose-control diet; ND, not done. *P ⬍ .01 vs. Dex-I/R by one-way ANOVA (Bonferroni). †P ⬍ .01 vs. Chol-I/R or Dex-I/R by one-way ANOVA (Bonferroni). ‡P ⬍ .05 vs. Dex-I/R by one-way ANOVA (Bonferroni).

Determination of CINC-1 Level in the Liver Homogenate. A small proportion of the liver tissue (approximately 400 mg) was homogenized with 4 volumes of RPMI-1640 medium in a Teflon-homogenizer for 15 seconds. The homogenate was immediately frozen in liquid nitrogen, then thawed at 37°C. After sonication for 10 seconds at 4°C the homogenate was centrifuged at 13,000g for 10 minutes at 4°C and the supernatant was used for determination of CINC-1 immunoreactive protein level with an ELISA kit (Amersham Pharmacia Biotech Inc.), which has a low detection limit of 5 pg/mL. Protein concentration was determined using Bradford’s reagent (Sigma Chemical Co., St. Louis, MO) and bovine serum albumin as standard. Statistical Analysis. All data are expressed as means ⫾ SEM. Difference between groups was evaluated using Bonferroni’s method after one-way ANOVA. In the case of histologic grading it was evaluated using Mann-Whitney’s U test. A P value less than .05 denoted the presence of a statistically significant difference.

RESULTS

however, a few layers of hepatocytes surrounding coagulative necrotic areas were shown to be TUNEL positive with dense condensation of nuclear chromatin in the livers of all groups of rats (Fig. 2A). In addition, TUNEL-positive hepatocytes were scattered in the liver, preferentially in the midzonal area (Fig. 2B). The number of the scattered cells with TUNELpositive nuclei was considerably high in both models of fatty liver, when compared with the nonfatty control liver. Because quantitation of TUNEL-positive cells was unreliable due to their clustered localization and coagulative necrosis, we attempted to evaluate the extent of apoptosis by detection of nucleosomal fragmentation as described below. Hepatic DNA Fragmentation After I/R in Fatty Livers. The levels of histone-associated oligonucleosomal DNA of the liver tissue at 4 hours after reperfusion were significantly higher in ethanol-fed animals (3-fold) and in cholesterol-fed animals

Hepatic Triglyceride and Cholesterol Contents, and Percents of Liver Weight over Body Weight. As shown in Table 1, hepatic

triglyceride contents were significantly increased in both fatty liver models when compared with the control. The triglyceride level in cholesterol-induced fatty livers was almost similar to that in ethanol-induced fatty livers. The liver was significantly enlarged in each of the fatty liver models as assessed by liver weight relative to body weight. Cholesterol content in cholesterol-induced fatty livers was significantly higher than that in alcohol-induced fatty or control livers. Hepatic I/R Injury and Neutrophil Accumulation. Hepatic I/R resulted in substantial liver injury in a time-dependent fashion as assessed by serum ALT level and hepatic histology. At 1 hour after reperfusion, serum ALT levels were slightly elevated, but histologically, no apparent necrosis was detected in any group, whereas pyknosis of nuclei and dissociation of hepatocytes were evident. Serum ALT levels increased progressively up to 4 hours after reperfusion, and the levels were less at 24 hours. The ethanol-fed group showed a significantly higher level of serum ALT (Table 2) and a significantly greater extent of hepatic necrosis than in the cholesterol-fed group or the control (Table 3). The number of neutrophils in the liver 4 hours after reperfusion was also significantly higher in the ethanol-fed rats than cholesterol-fed group or the control (Fig. 1). In Situ TUNEL Staining in Livers. A very few hepatocytes (less than 1 out of 200 hepatocytes) were shown to have TUNELpositive nuclei in sections of livers subjected to a sham operation or 1 hour reperfusion. At 4 hours after reperfusion,

FIG. 3. Hepatic DNA fragmentation at 4 hours after ischemia/reperfusion. Lane 0, negative control (normal rat liver); lanes 1 and 4, ethanol-fed rats; lanes 2 and 5, cholesterol-fed rats; lanes 3 and 6, dextrose-fed control rats (detected by autoradiogram as described in the Materials and Methods). Free 32P-␣-CTP was detected below the low-molecular DNA fragment (180 bp). Representative data from 2 of 6 experiments.

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FIG. 4. EMSA for NF-␬B binding activity (indicated by the arrows). Gel electrophoresis of CINC–NF-␬B motif/protein complexes in liver nuclear extracts (4 ␮g) from a representative untreated rat (lane 1), sham-operated rats (lanes 2, 6, and 9), and animals subjected to 30 minutes of hepatic ischemia and 1 hour (lanes 3, 4, 7, and 10) and 24 hours (lanes 5, 8, and 11) of reperfusion. Lanes 1-5, ethanol-fed rats; lanes 6-8, cholesterol-fed rats; lanes 9-11, control rats. Alc, ethanol-fed; Chol, cholesterol-fed; and Dex, dextrose-fed control. At 1 hour after I/R, the relative increase (over sham-operated animals) in NF-␬B binding activity of liver nuclear extracts from ethanol-fed, cholesterol-fed, and control rats was 5.20 ⫾ 0.94-fold (n ⫽ 4), 2.52 ⫾ 0.32-fold (n ⫽ 4) and 2.88 ⫾ 0.57-fold (n ⫽ 4), respectively, as assessed by densitometric analysis (P ⬍ .02, Alc vs. Chol; P ⬍ .05, Alc vs. Dex, by one-way ANOVA). To evaluate the specificity of NF-␬B binding, lanes 13 to 16 depict binding of nuclear extracts from a representative ethanol-fed rat at 1 hour of reperfusion. Lane 12, no extract; lane 13, nuclear extract only; lane 14, competition of NF-␬B binding in the same extract with a 200⫻ molar excess of cold CINC–NF-␬B oligo; lane 15, supershift assay using 1 ␮L of antibody against p50; lane 16, supershift assay using 1 ␮L of antibody against p65. Representative data from 4 experiments.

(2.5-fold) when compared with the control (Table 4). These levels diminished to near the baseline at 24 hours in cholesterol-fed animals. To confirm the difference in hepatic DNA fragmentation quantified by ELISA, we performed agarose gel electrophoresis of DNA isolated from the liver after radiolabeling the DNA with the Klenow fragment to enhance the detection sensitivity. The characteristic ladder pattern of internucleosomal DNA cleavage into 180-bp fragments was observed at 4 hours after reperfusion. Furthermore, the degree of DNA cleavage was considerably enhanced in both models of fatty liver versus nonfatty control liver (Fig. 3). NF-␬B and AP-1 Activation After I/R in Fatty Livers. EMSA performed using liver nuclear extract and a CINC–NF-␬B or an AP-1 oligonucleotide showed low basal levels of binding activity in preparations obtained from sham-operated rats (Figs. 4 and 5). One hour of reperfusion activated the binding activity, which persisted, although at a reduced level, at 24 hours after reperfusion. At 1 hour after I/R, ethanol-fed animals showed a significantly high NF-␬B binding activity with a mean 5.2-fold (n ⫽ 4) increase over the sham-operated group when compared with each of the other groups (2.5-fold in-

crease in the cholesterol-fed group; 2.9-fold in the control group, n ⫽ 4) (Fig. 4). Supershift analysis revealed that p50 and p65 were involved in the activation of binding on the NF-␬B consensus site. In contrast, AP-1 binding activity at 1 hour was considerably enhanced in both fatty liver models, when compared with the control (5.3-fold [over the shamoperated group] in the ethanol-fed group; 7.0-fold in the cholesterol-fed group; 3.0-fold in the control, n ⫽ 4) (Fig. 5). Specificity of the binding was shown by competition with a 200-fold amount of the same unlabeled oligonucleotide, and by the lack of competition with an unrelated oligonucleotide. Supershift analysis showed that at least c-jun might be included in the AP-1 DNA complex at 1 hour after reperfusion. Inflammatory Cytokine and Neutrophil Chemokine mRNA Expression in the Liver. To evaluate the downstream transcriptional

effects of NF-␬B, we quantitated the expression of neutrophil chemokine and inflammatory cytokine mRNAs in the liver by using RT-PCR and Northern blotting. CINC-1 mRNA level as assessed by RT-PCR in the liver of ethanol-fed rats was significantly higher than the control at 1 hour and than other groups at 4 hours after I/R. The levels were reduced to the

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FIG. 5. EMSA for AP-1 binding activity (indicated by arrows). Gel electrophoresis of AP-1 motif/protein complexes in the same liver nuclear extracts as those used in Fig. 4 from a representative untreated rat (lane 1), sham-operated rats (lanes 2, 6, and 9), and rats subjected to 30 minutes of hepatic ischemia and 1 hour (lanes 3, 4, 7, and 10) and 24 hours (lanes 5, 8, and 11) of reperfusion. Lanes 1-5, ethanol-fed rats; lanes 6-8, cholesterol-fed rats; lanes 9-11, control rats. Alc, ethanol-fed; Chol, cholesterol-fed; and Dex, dextrose-fed control. At 1 hour after I/R, the relative increase (over sham-operated animals) in AP-1 binding activity of liver nuclear extracts from ethanol-fed, cholesterol-fed, and control rats was 5.33 ⫾ 1.69-fold (n ⫽ 4), 6.96 ⫾ 0.97-fold (n ⫽ 4) and 2.97 ⫾ 1.06-fold (n ⫽ 4), respectively, as assessed by densitometric analysis (P ⫽ .22, Alc vs. Dex, P ⫽ .055, Chol vs. Dex, by one-way ANOVA). To evaluate the specificity of AP-1 binding, lanes 13 to 19 depict bindings of nuclear extracts from a representative ethanol-fed rat at 1 hour of reperfusion. Lane 12, no extract; lane 13, nuclear extract only; lane 14, competition of AP-1 binding in this same extract with a 200⫻ molar excess of cold AP-1 oligo; lane 15, the same extract with a 200⫻ molar excess of cold CINC–NF-␬B oligo; lanes 16 and 17, supershift assay using 0.5 and 1 ␮L, respectively, of antibody against c-fos; lanes 18 and 19, supershift assay using 0.5 and 1 ␮L, respectively, of antibody against c-jun. Representative data from 4 experiments.

baseline at 24 hours (Fig. 6). Northern blotting analysis confirmed the significant difference in CINC-1 mRNA expression between ethanol-fed rats and each of the other groups at 4 hours, although 2 distinct signals were detected (Fig. 7). The immunoreactive CINC-1 protein level in the liver was also significantly higher in ethanol-fed rats than in each of the other groups at 4 hours after reperfusion (P ⬍ .05, by one-way ANOVA). The CINC-1 protein levels were 101 ⫾ 22.3 (pg/mg protein) in ethanol-fed rats versus 58.4 ⫾ 9.68 in cholesterolfed rats and 48.8 ⫾ 7.88 in the control rats (n ⫽ 6 in each group). The elevated levels of CINC-1 protein at 4 hours paralleled both increased CINC-1 mRNA levels and NF-␬B binding activity. Finally, we determined hepatic mRNA levels of other inflammatory cytokines that are related to NF-␬B activity. In ethanol-fed rats, warm I/R resulted in a rapid increase in hepatic mRNA levels of IL-1␣ (with a peak level recorded at 1 hour) and TNF-␣ (with a peak level at 4 hours) (Fig. 8). Ethanol-fed rats showed a significantly higher expression of IL-1␣ and tended to show a high expression of TNF-␣ mRNA in the liver as shown in Fig. 8. DISCUSSION

Clinically, fatty liver is usually seen in patients with longterm ethanol consumption and/or obesity. Most studies investigating the effects of fatty liver on hepatic injury after I/R or transplantation have used rats fed a choline-deficient diet.39-42 In such a model, however, fatty liver results from malnutrition mainly caused by an impairment of lipoprotein excretion from hepatocytes. We induced alcoholic and nonalcoholic fatty liver by the isocaloric feeding formula, as described in the

Materials and Methods, to simulate a hypernutrition-related fatty liver as well as to distinguish experimentally ethanolselective phenomena from those associated commonly with fatty liver. An important finding in the present study is that severe hepatic necrosis is associated with apoptosis after I/R only in alcoholic fatty liver, in which neutrophils are recruited and activated after I/R. The microenvironment generated by inflammatory cytokines such as CINC-1 or TNF-␣ via NF-␬B activation might be crucial for the apoptosis-associated necrosis. It is possible that our results may reflect only a quantitative difference in a susceptibility to I/R between the 2 fatty liver models, because a longer period of ischemia has been reported to cause extensive apoptosis and necrosis even in normal rats.43,44 We decided to perform a 30-minute period of ischemia for the following reasons; a 30-minute period of ischemia has been generally considered reversible for a normal rat liver,45 and in a series of our preliminary experiments a 60-minute period of ischemia resulted in a failure of homogenous reperfusion and in animal death by 24 hours after I/R in some rats with fatty livers. Our results showed that hepatic apoptosis at 4 hours after I/R increased to a similar degree in each of the 2 fatty liver models in rat models (alcoholic and nonalcoholic) compared with the control. It is now generally accepted that apoptosis occurs in many pathologic as well as physiologic processes. There are 2 concepts on a pathologic relationship between apoptosis and necrosis, i.e., (1) they are distinct forms of cell death, thus each of them is independent of the other; and (2) apoptosis positively contributes to necrosis. From the former

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moxic and anoxic regions. To strengthen the evidence for apoptosis and to quantify its extent, we determined histoneassociated oligonucleosomal level as well as electrophoretic analysis for DNA fragmentation. Persistent increase in hepatic histone-associated oligonucleosomal level at 24 hours after I/R in alcoholic fatty liver suggests that apoptosis may contribute to the remodeling process during the recovery from necrosis, as reported previously.48 The two transcription factors, NF-␬B and AP-1, are known to be activated after I/R19-21 and cooperatively induce the expression of cytokines contributory to neutrophil-mediated inflammation.50 NF-␬B activity is thought to be regulated by a redox state. Long-term ethanol intake potentiated the production of reactive oxygen species in the liver after I/R,24 which may in turn enhance NF-␬B binding activity. Our results suggested that NF-␬B activation following I/R contributes to the enhanced production of a proinflammatory cytokine and chemokine that leads to hepatic necrosis in ethanol-fed rats. On the other hand, the activation of AP-1 is likely to be related to hypoxia during the ischemic phase.51,52 This fact may account

FIG. 6. Effects of alcoholic or nonalcoholic fatty liver on CINC-1 mRNA expression in the rat liver at 4 hours after the sham operation and at 1, 4, and 24 hours after I/R.(A) CINC-1 mRNA expression was determined by RT-PCR from hepatic RNA isolated as described in the Materials and Methods. Al, ethanol-fed; Ch, cholesterol-fed; Dx, dextrose-fed control. Data represent means ⫾ SEM (n ⫽ 3-6). #P ⬍ .01 vs. sham or 24 hours, by two-way ANOVA. *P ⬍ .05 vs. Dx; **P ⬍ .01 vs. Ch or Dx by one-way ANOVA (Bonferroni). (B) Amplification of hepatic CINC-1 and ribosomal S6 protein mRNA transcripts by RT-PCR from ethanol-fed (lanes 1-5), cholesterol-fed (lanes 6-9), and control rats (lanes 10-13). MW, molecular weight markers (100-bp DNA ladder); lane 1, an untreated rat liver; lanes 2, 6, and 10, sham-operated rat livers; lanes 3, 7, and 11, at 1 hour after reperfusion; lanes 4, 8, and 12, at 4 hours after reperfusion; lanes 5, 9, and 13, at 24 hours after reperfusion; lane 14, negative (normal rat liver) control. PCR products were 419 bp in size for ribosomal protein S6 and 223 bp for CINC-1, respectively.

point of view, coexistence of both types of cell death in a pathologic situation may be accounted for by the fact that individual cell death is decided by the availability of adenosine triphosphate.46 Recently, however, some evidences in the in vivo studies using caspase inhibitors have supported a hypothesis that apoptosis constitutes a potential trigger of inflammation leading to cell necrosis in the liver or the kidney after I/R.43,47 Our present results showed that apoptosis does not always lead to necrosis in the liver following I/R, indicating that apoptosis per se does not cause inflammation even in a pathologic situation. Evidence for the involvement of apoptosis is provided by the presence of TUNEL-positive hepatocytes and low-molecular DNA fragmentation. TUNEL-positive hepatocytes surrounding the necrotic foci indicate that apoptosis is associated with an inflammatory necrotic process. The characteristic localization of hepatocytes with apoptotic nuclei, including those scattered preferentially in the midzonal area, is consistent with previous findings.48,49 Marotto et al.49 observed that cell death during low-flow hypoxia occurred earliest in the midzonal area, or at the anoxic edge, i.e., the border of nor-

FIG. 7. Effects of alcoholic or nonalcoholic fatty liver on CINC-1 mRNA expression in the rat liver at 4 hours after ischemia/reperfusion. (A) Quantitation of CINC-1 mRNA by Northern analysis. The ratios of CINC-1 mRNA over 28S rRNA are represented. The ratios were calculated from densitometric analysis. Data represent means ⫾ SEM (n ⫽ 5). *P ⬍ .01 vs. Chol or Dex, by one-way ANOVA (Bonferroni). Alc, ethanol-fed; Chol, cholesterol-fed; Dex, dextrose-fed control. (B) Representative Northern blot analysis of total RNA (20 ␮g) extracted from rat livers at 4 hours after ischemia/reperfusion. Lanes 1-5, ethanol-fed; lanes 6-8, cholesterol-fed; lanes 9-10, dextrose-fed control rats. 18S and 28S correspond to 18S and 28S rRNA, respectively.

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FIG. 8. Effects of alcoholic or nonalcoholic fatty liver on the expression of IL-1␣ and TNF-␣ mRNAs in the rat liver at 1, 4, and 24 hours after I/R. (A) IL-1␣ and TNF-␣ mRNA expression was determined by RT-PCR from hepatic RNA isolated as described in the Materials and Methods. Data represent means ⫾ SEM (n ⫽ 3-6). Al, ethanol-fed; Ch, cholesterol-fed; and Dx, dextrose-fed control. #P ⬍ .025 vs. sham, ##P ⬍ .01 vs. sham, 1 hour and 24 hours, by two-way ANOVA. *P ⬍ .08 vs. Dx, **P ⬍ .02 vs. Ch or Dx, by one-way ANOVA (Bonferroni). (B) Amplification of hepatic IL-1␣ and TNF-␣ mRNA transcripts by RT-PCR, using the same cDNA used in Fig. 6, from ethanol-fed (lanes 1-5), cholesterol-fed (lanes 6-9), and control rats (lanes 10-13). MW, molecular weight markers (100-bp DNA ladder); lane 1, liver of untreated rat; lanes 2, 6, and 10, liver of sham-operated rat; lanes 3, 7, and 11, at 1 hour after reperfusion; lanes 4, 8, and 12, at 4 hours after reperfusion; lanes 5, 9, and 13, at 24 hours after reperfusion; lane 14, negative control (normal rat liver). PCR products were 297 bp in size for IL-1␣ and 269 bp for TNF-␣, respectively.

for a tendency of AP-1 activity to increase in both fatty liver models versus the control liver. The significance of NF-␬B and AP-1 activation in such an acute reaction, however, needs to be cautiously evaluated, because these activities are considered to be related to stress phenomena in a nonspecific manner,53 or they might positively regulate the expression of the immediate-early gene to prepare the future proliferative response needed after cell necrosis.54 In this study, high levels of immunoreactive CINC-1 were associated with increase of CINC-1 mRNA and NF-␬B binding activity in ethanol-fed rats. The CINC-1 mRNA levels determined by Northern blotting paralleled those by RT-PCR. The 2 detected bands were probably accounted for by an alternative splicing of CINC-1 mRNA. The possibility was not completely excluded that 1 of the 2 signals was derived from some other members of CINC family such as CINC-2␣ or -2␤, which have a highly conserved sequence domain.55,56 Al-

though TNF-␣ acts as a strong activator of NF-␬B,57,58 the weak TNF-␣ mRNA expression at 1 hour, when NF-␬B was already activated, suggests that NF-␬B is rather responsible for I/R-induced increases in TNF-␣ as well as CINC-1 transcription. IL-1␣ mRNA levels at 1 hour in the present study paralleled NF-␬B activity, suggesting that IL-1␣, in addition to reactive oxygen species, may be responsible for NF-␬B activation. In summary, the alcoholic fatty liver exposed to I/R showed higher NF-␬B activity and induction of inflammatory cytokines as well as CINC-1 followed by hepatic necrosis with neutrophil infiltration as alcohol-selective phenomena, in comparison with a nonalcoholic fatty liver model. The proinflammatory state in alcoholic-fatty liver may be attributed to activated hepatic macrophages in ethanol-fed rats as previously shown.17,59 The effects of inhibition of hepatic macrophage or neutrophil activity on the I/R liver injury warrant further investigation.

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