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Role of adhesion molecules in the development of massive hepatic necrosis in rats
Role of Adhesion Molecules in the Development of Massive Hepatic Necrosis in Rats SATOSHI MOCHIDA,1 AKIHIKO OHNO,2 MASAHIRO ARAI,2 TAKUYA TAMATANI,3 MASAYUKI MIYASAKA,4 1 AND KENJI FUJIWARA
Massive hepatic necrosis develops after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes as a result of microcirculatory disturbance caused by endothelial cell destruction by activated macrophages in the hepatic sinusoids. Immunohistochemical hepatic expression of intercellular adhesion molecule–1 (ICAM-1) and lymphocyte function– associated antigen 1a (LFA-1a) and the effect of monoclonal antibodies against both adhesion molecules on liver necrosis provoked after endotoxin administration was studied in these rats. There were increased stains of ICAM-1 in endothelial cells and LFA-1a in macrophages in the hepatic sinusoids in Propionibacterium acnes–pretreated rats compared with normal rats. Such stains were further increased soon after endotoxin administration, followed by development of hepatic necrosis. Monoclonal antibodies against both adhesion molecules significantly attenuated the extent of liver injury compared with controls, without affecting the infiltration and activation of hepatic macrophages. Polyclonal antibodies against polymorphonuclear leukocytes eradicated circulating neutrophils, but did not change such liver injury, although gum arabic, which suppressed macrophage activation, attenuated the extent of liver injury. Thus, adhesion between endothelial cells and activated macrophages in the hepatic sinusoids via ICAM1 and LFA-1a is essential for the initiation of massive hepatic necrosis of this type. Contribution of neutrophils seems less likely. (HEPATOLOGY 1996;23:320-328.)
Cell-to-cell interaction through adhesion molecules has attracted attention in the pathogenesis of various organ injuries.1-5 Treatments using antibodies against intercellular adhesion molecule 1 (ICAM-1) and lym-
Abbreviations: ICAM-1, intercellular adhesion molecule 1; LFA-1a, lymphocyte function–associated antigen 1a; PBS, phosphate-buffered saline; Ig, immunoglobulin; NBT, nitro blue tetrazolium; PMA, phorbol 12-myristate 13acetate; TNF, tumor necrosis factor; ALT, alanine transaminase. From the 1Third Department of Internal Medicine, Saitama Medical School, Saitama, Japan; 2First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan; 3Japan Tabacco Inc., Pharmaceutical Basic Research Center, Kanagawa, Japan; and 4Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Osaka, Japan. Received March 8, 1995; accepted September 7, 1995. Address reprint requests to: Kenji Fujiwara, M.D., Ph.D., Third Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyamacho, Iruma-gun, Saitama 350-04, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2302-0018$3.00/0
phocyte function–associated antigen 1a (LFA-1a) demonstrated the contribution of these adhesion molecules to induction of arthritis by adjuvant in rats1 and acute rejection after cardiac transplantation in mice.5 Adhesion molecules also seem to play a role in the development of liver injury, as the expression of several adhesion molecules in human liver has been reported to increase in association with liver diseases.6-11 Our previous observations demonstrate that fibrin deposition due to endothelial cell destruction in the hepatic sinusoids causes massive hepatic necrosis,12,13 and such endothelial cell destruction can be produced by activated hepatic macrophages in massive hepatic necrosis induced by endotoxin in rats pretreated with heat-killed Propionibacterium acnes (P. acnes; Corynebacterium parvum).14-19 With isolated perfused liver system of this model, activated hepatic macrophages are proven to destroy sinusoidal endothelial cells but not hepatocytes.19 However, the reason why activated macrophages exclusively attack endothelial cells in the hepatic sinusoids despite the fact that macrophages can damage hepatocytes in vitro20-22 is to be elucidated. Also, the contribution of other cells to massive hepatic necrosis is important, because cytotoxic cells such as neutrophils are circulating in vivo.1,2,23-26 ICAM-1 and its ligand, LFA-1a, have been reported to be expressed in isolated endothelial cells and macrophages, respectively.27,28 This prompted us to investigate the role of both adhesion molecules in the interaction between endothelial cells and activated macrophages in the hepatic sinusoids in massive hepatic necrosis of this type and the contribution of neutrophils as they are shown to express LFA-1a as well.29 MATERIALS AND METHODS Experimental Designs Male Fisher rats (Charles River Japan, Atugi, Japan) weighing 170 to 190 g were maintained on a commercial pelleted diet and water ad libitum in a room at 22 { 27C under normal laboratory lighting conditions. All animal study protocols conformed to National Research Council criteria for humane care. Experiment I. Eighteen rats received 15 mg/kg body weight heat-killed P. acnes (C. parvum whole cells: Ribi Immunochem Research Inc., Hamilton, MO) as a 7.5 mg/mL suspension in saline through the femoral vein. On the 7th day, they were given an intravenous injection of 200 mg/kg endotoxin (lipopolysaccharide from Escherichia coli 026:B6;
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Difco, Detroit, MI) as a 100 mg/mL solution in saline, and were killed 0, 3, or 5 hours later for immunohistochemical examination of the liver on light or electron microscopy. Six normal rats were also killed for a similar examination. Experiment II. Eighteen rats received a daily intraperitoneal injection of 1 mg/kg body weight of anti-rat ICAM-1 and/ or LFA-1a monoclonal antibodies as 1 mg/3 mL solutions in phosphate-buffered saline (PBS) for 10 days (ICAM-1 and LFA-1–, and ICAM-1–, and LFA-1–treated groups). Another 12 rats were given similarly 1 or 2 mg/kg body weight of nonimmune mice immunoglobulin (Ig)G as a 1 mg/3 mL solution in PBS (control group). All rats received heat-killed P. acnes on the 4th day and endotoxin 24 hours after the final antibody treatment. For the determination of the extent of liver injury, blood was collected through the inferior caval vein under anesthesia with ether 5 hours after endotoxin injection, with an empty plastic syringe and a plastic syringe containing 1:10 volume of 3.8% sodium citrate solution to prepare serum and plasma, respectively, and the liver was excised. Experiment III. Twenty rats received both antibodies or nonimmune mice IgG and heat-killed P. acnes similarly. Eight rats were subjected to liver perfusion with nitro blue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) 24 hours after the final antibody treatment for the evaluation of the activation and infiltration of hepatic macrophages. Another 12 rats received an intravenous injection of endotoxin 24 hours after the final antibody treatment, and 1 hour later blood was collected through the inferior caval vein under anesthesia with ether for the determination of serum tumor necrosis factor-a (TNFa) concentration. Experiment IV. Twelve rats received heat-killed P. acnes similarly. On the 6th day, They were given an intravenous injection of 0.2 mL/kg rabbit antisera against rat polymorphonuclear leukocytes (Inter-Cell Technologies, Inc., Somerville, NJ) (antisera-treated group) or the same volume of normal rabbit sera (control group) as 10% solution in saline. Twenty-four hours later, 0.6 mL blood was collected through the femoral vein using a plastic syringe containing 60 units of heparin (Novo Industri A/S, Copenhagen, Denmark) for the count of peripheral neutrophils, and then the rats were given an intravenous injection of endotoxin similarly. Five hours after endotoxin administration, blood was collected through the inferior vena cava for the measurement of the parameters of liver injury under anesthesia with ether, and the liver was excised for the histological examination. Experiment V. Eighteen rats received a daily intraperitoneal injection of 100 mg gum arabic (Sigma Chemical Company, St. Louis, MO) as 10% solution in saline (gum arabic– treated group) or the same volume of saline (control group) for 10 days. Six rats in both groups were given heat-killed P. acnes and endotoxin on 4th and 11th days, respectively. Five hours after endotoxin administration, blood was collected through the inferior vena cava for the determination of the extent of liver injury under anesthesia with ether, and the liver was excised for the histological examination. Another three rats in both groups were given heat-killed P. acnes on the 4th day, and they were subjected to liver perfusion with NBT and PMA on the 11th day for the evaluation of hepatic macrophage activation. Preparation of Anti-rat ICAM-1, LFA-1a Monoclonal Antibodies and Nonimmune Mice IgG Monoclonal antibodies used were anti-rat ICAM-1, 1A29 (mouse IgG1)30 and anti-rat LFA-1a, WT1 (mouse IgG2a).31 Hybridoma cells that produce both monoclonal antibodies
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were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 10% fetal bovine serum (Gibco Laboratories, Life Technologies Inc., Grand Island, NY) for several days. Then cultured hybridoma cells were injected into the abdominal cavity of female Balb/c nude mice (Charles River Japan) pretreated with 0.5 mL of 2,6,10,14-tetramethyl-pentadecane intraperitoneally, and ascites were collected 7 days later. Both antibodies were purified from the ascites using protein G affinity column (Protein G Sepharose 4 Fast Flow; Pharmacia, Uppsala, Sweden), and concentrated using ultrafiltration membrane (AMICON; W.R. Grace & Co., Danver, MA).5 Control IgG was purified similarly from nonimmune mice serum (Chemicon International Inc., Temecula, CA). Immunohistochemical Study of the Liver Under anesthesia with an intraperitoneal injection of 50 mg/kg pentobarbital sodium, the abdomen was cut open. For light microscopic examination, the liver was perfused by the portal vein with saline at a flow rate of 10 mL/minute for 1 minute, and excised. Blocks of 5 mm3 cut off with a blade from excised livers were immersed into ornithine-carbamoyltransferase Compound (Miles Inc., Elkhart, IN), snap-frozen in liquid nitrogen, and stored at 0807C. From frozen blocks, 5-mm cryostat sections were made and fixed in absolute acetone for 10 minutes. Endogenous peroxidase activity was blocked in methanol containing 0.3% hydrogen peroxide for 15 minutes, and nonspecific binding sites in whole rabbit serum for 20 minutes. Then a two-step indirect immunoperoxidase procedure was performed; sections were incubated with anti-rat ICAM-1 or LFA-1a monoclonal antibody as a solution of 1 mg/mL in PBS for 1 hour, and then with peroxidase-conjugated rabbit anti-mouse IgG (working dilution 1:100, Vector Laboratories, Inc., Burlingame, CA) for 1 hour at room temperature. The reaction product was developed by incubation for 5 minutes in solution of 0.05% 3,3*-diaminobenzidine, pH 7.2, containing 0.01% hydrogen peroxide (Vector Laboratories). Counterstaining was done in 4% methyl green solution for 2 minutes. In controls, nonimmune mouse IgG was used instead of primary antibodies. For electron microscopic examination, the liver was perfused with saline in the same manner as for light microscopic examination, and with cold fixative containing 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mol/L PBS. Three blocks of 5 mm3 cut off with a blade from the excised liver were immersed and shaken in PBS containing 5% sucrose, and successively in PBS containing 10%, 20%, 30%, and 40% sucrose at 47C. The blocks were then put into ornithine-carbamoyltransferase (OCT) compound and rapidly frozen in dry ice–acetone. Eight-micrometer cryostat sections were made from frozen blocks. A two-step indirect immunoperoxidase procedure was performed in the same way as for light microscopic examination. After fixation of the sections in 2.5% glutaraldehyde solution, the reaction product was developed by incubation in a 0.05% dimethylaminoazobenzene solution without H2O2 and then in dimethylaminoazobenzene solution containing 0.005% H2O2 . The sections were postfixed in 1% osmium tetroxide and dehydrated through graded concentrations of ethanol. Epoxy resin in gelatin capsules was placed on the section at 607C. Ultrathin sections made with a glass knife were observed under Hitachi (Hitachi, Tokyo, Japan) H-800 electron microscope at 100 kV. Procedure for Liver Perfusion With Nitro Blue Tetrazolium and Phorbol Myristate Acetate As previously described,32,33 after injection of 200 units of heparin into the inferior vena cava under anesthesia with an
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TABLE 1. Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Liver Function Tests and Histological Extent of Liver Injury 5 Hours After Endotoxin Administration in P. acnes–Pretreated Rats (Experiment II)
Groups
Control (IgG: 2 mg/kg) ICAM-1– and LFA-1a –treated Control (IgG: 1 mg/kg) ICAM-1–treated LFA-1a –treated
Serum ALT (KU)
450.8 175.0 662.9 666.7 326.7
{ { { { {
196.7† 110.0§ 380.5 260.7 220.4
Prothrombin Time (s)
Plasma Antithrombin III (%)
0
1
2
3
22.7 15.6 22.8 20.8 21.2
60.2 75.2 52.3 49.2 48.3
0 1 0 0 0
1 4 0 0 0
1 1 0 0 1
4‡ 0Ø 6 6 5
{ { { { {
4.5 2.2\ 4.7 3.0 4.8
{ { { { {
10.3 8.9§ 6.4 5.2 11.2
Extent of Liver Injury* (Grade)
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as follows: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering õ5%; grade 2, 5% to 10%; grade 3, ú10%. † Values given as mean { SD of six rats. Normal values of serum ALT, prothrombin time, and plasma antithrombin III are 22 { 6 KU, 10.1 { 0.6 seconds, and 105.0% { 2.7%, respectively (n Å 4). ‡ No. of rats. § P õ .05. \ P õ .01 vs. control (IgG: 2 mg/kg) group, by Student’s t test. Ø P õ .05 vs. control (IgG: 2 mg/kg) group, by Mann-Whitney test.
intraperitoneal injection of pentobarbital sodium, the liver was perfused successively with Ca2/- and Mg2/-free Hanks’ balanced salt solution (Gibco Laboratories) for 5 minutes, Dulbecco’s minimal essential medium (Nissui Pharmaceutical) with 0.5 mg/mL PMA (Sigma Chemical Company) for 5 minutes, 0.05% NBT (Grade III, Sigma Chemical Company) in Eagle’s minimal essential medium with PMA for 10 minutes, and finally with Hanks’ balanced salt solution for 5 minutes. The perfusion was performed at a flow rate of 20 mL/min at 377C with continuous O2 bubbling. All the perfusates contained 20 mmol/L HEPES, pH 7.4. Then the excised liver was fixed in formalin and embedded in paraffin. Evaluation of the Extent of Liver Injury The extent of liver injury was assessed from serum alanine transaminase (ALT) activity, plasma antithrombin III activity, and prothrombin time as well as from the histological extent of liver necrosis. Serum ALT activity was determined using a commercial kit (Iatron Laboratories Inc., Tokyo, Japan). Prothrombin time was measured in plasma using SIMPLASTIN (Division of Warner Lambert Co., Cockeysville, NJ) and a fibrometer (Division of Beckton, Dickinson and Company, Cockeysville, MD). Plasma antithrombin III concentration was determined with Coatest Antithrombin III kit (Kabi Diagnostica, Stockholm, Sweden). Histological examination was performed using formalinfixed and paraffin-embedded liver specimens. All slides were evaluated at random by one of the authors on light microscopy. The extent of liver necrosis was classified using a four-
degree score as shown in the footnote of Table 1 with sections counterstained with hematoxylin-eosin. Evaluation of the Infiltration and Activation of Hepatic Macrophages The activation of hepatic macrophages was based on their stimulatory stage determined by the extent of formazan deposition on sections of the liver after perfusion with NBT and PMA, which were counterstained with Kernechtrot as previously reported.32-37 The infiltration of macrophages into the liver was estimated from liver weights and the size and number of granulomas on the same sections of the liver after perfusion with NBT and PMA counterstained with Masson’s trichrome, as detailed in the footnote of Table 2. All slides were evaluated at random by one of the authors on light microscopy. The activation and infiltration of hepatic macrophages were also estimated from serum concentration of TNFa using a commercial enzyme-linked immunosorbent assay kit (Factor Test Mouse TNF-a kit: Genzyme Diagnostics, Boston, MT). Count of Peripheral Neutrophils The number of total peripheral white blood cells was counted by light microscopy using a Neubauer hemocytemeter after diluting the heparinized blood to 1:10 in volume in Turk’s fluid. Peripheral neutrophil count was calculated from this number and the relative number of neutrophils to white blood cells, which was determined on blood smear stained by Wright and Giemsa.
TABLE 2. Effect of Anti-Rat ICAM-1 and LFA-1a on Infiltration and Activation of Hepatic Macrophages 7 Days After P. acnes Administration in Rats (Experiment III) Granuloma* Groups
Liver Weight (% Body Weight)
No.
Size (mm)
Serum TNFa Concentration (pg/mL)
Control ICAM-1– and LFA-1a –treated
5.73 { 0.33† 5.63 { 0.40
115.0 { 22.7 118.3 { 23.2
57.4 { 5.2 64.5 { 5.0
1,097 { 407‡ 1,463 { 454
* The number of granulomas is total numbers counted from five fields randomly selected under magnification at 100X. The size is the mean of largest diameter of 30 granulomas randomly selected under magnification at 200X. † Values given as mean { SD of four rats. Normal liver weight is 4.26% { 0.45% of body weight (n Å 4). ‡ Values given as mean { SD of six rats.
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FIG. 1. Light micrographs showing immunohistochemical expression of ICAM-1 and LFA-1a in the liver in normal and Propionibacterium acnes (P. acnes)–treated rats (experiment I). (A) ICAM-1 staining in normal liver. (Original magnification 1204.) Very slight stain is seen in sinusoidal cells throughout the liver. (B) LFA-1a staining in normal liver. (Original magnification 1270.) Faint stain is observed in sinusoidal cells in the periportal area (arrows). (C) ICAM-1 staining in P. acnes–treated liver. (Original magnification 1326.) Moderate stain is seen in sinusoidal cells throughout the liver, and marked stain in granulomas (arrowhead). (D) LFA-1a staining in P. acnes–treated liver. (Original magnification 1270.) Faint stain is observed in sinusoidal cells throughout the liver (arrows), and marked stain in granulomas. (E) ICAM-1 staining in the liver 3 hours after endotoxin treatment after pretreatment with P. acnes. (Original magnification 1326.) Marked stain is seen in sinusoidal cells and granulomas (arrowhead) throughout the liver, but not in hepatocytes. (F) LFA-1a staining in the liver 3 hours after endotoxin treatment after pretreatment with P. acnes. (Original magnification 1326.) Marked stain is seen in sinusoidal cells (arrows) and granulomas (arrowhead) throughout the liver.
RESULTS Immunohistochemical Expression of ICAM-1 and LFA-1a in Rat Livers (Experiment I)
Immunohistochemical findings of ICAM-1 and LFA1a in the liver were not so different among three rats in each group. Controls of these adhesion molecules showed no staining. In normal liver, sinusoidal cells showed slight ICAM1 stains throughout the liver and LFA-1a stains in the periportal area on light microscopy (Fig. 1A and B). Electron microscopic examination demonstrated that sinusoidal cells with such slight ICAM-1 and LFA-1a staining were endothelial cells and Kupffer cells, respectively. P. acnes–treated rats showed scattered granulomas and infiltrating macrophages in the hepatic sinusoids on the 7th day and stains of ICAM-1 and LFA-1a in sinusoidal cells throughout the liver more prominently compared with normal rats (Fig. 1C and D). Such stains of both adhesion molecules were further increased 3 hours after endotoxin administration, but hepatic necrosis was not present. Both adhesion molecules were also stained in granulomas, but not in hepatocytes (Fig. 1E and F ). Electron-microscopic examination showed that sinusoidal cells with such
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marked ICAM-1 and LFA-1a staining were endothelial cells and macrophages, respectively (Fig. 2A and B). Coagulative necrosis was observed in the midzonal areas 5 hours after endotoxin administration. In these rats, marked stains of ICAM-1 and LFA-1a were found in sinusoidal endothelial cells and hepatic macrophages, respectively, throughout nonnecrotic areas. Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment II)
As shown in Table 1, endotoxin administration produced increased serum ALT activity, prolonged prothrombin time and decreased plasma AT III concentration 5 hours later in P. acnes–pretreated rats. These derangements were significantly attenuated by treatment with both anti–ICAM-1 and LFA-1a antibodies. Such derangements, however, were not attenuated by the treatment with anti–ICAM-1 or LFA-1a antibody alone. On liver histology, all rats showed coagulative necrosis in the midzonal areas in the control groups; four of six rats and all of six rats showed the necrosis of grade 3 in 2 mg/kg and 1 mg/kg control groups, respectively.
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FIG. 2. Electron micrographs showing immunohistochemical expression of ICAM-1 and LFA-1a in the liver 3 hours after endotoxin administration in P. acnes–treated rats (experiment I). (A) ICAM-1. Sinusoidal endothelial cells show ICAM-1 stains along with the membrane of the cells. There is no stain in hepatocytes. (Original magnification 111,200.) (B) LFA-1a. Staining can be seen on the membrane of hepatic macrophage. Hepatocyte and endothelial cells show no stain. (Original magnification 113,800.)
In contrast, five of six rats showed the necrosis of grade 0 or 1 in the treatment group with both antibodies. However, treatment with anti–ICAM-1 or LFA-1a antibody alone did not affect the histological extent of liver injuries.
On liver histology, all rats showed coagulative necrosis of grade 2 or 3 in the midzonal areas. Administration of the antisera affected neither the deranged liver function tests nor the histological extent of liver injury in P. acnes– and endotoxin-treated rats.
Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Infiltration and Activation of Hepatic Macrophages in P. acnes–Treated Rats (Experiment III)
Effect of Gum Arabic on Activation of Hepatic Macrophages and Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment V)
Formazan deposition in hepatic macrophages was not so different among three rats in both control and antibody-treated groups. As shown in Table 2, infiltration of macrophages into the liver was remarkable 7 days after P. acnes administration in the control group, because liver weight and the number and size of hepatic granulomas were significantly increased compared with normal rats. Those macrophages were prominently activated, because they contained marked formazan deposition (Fig. 3A). Treatment with both antibodies affected neither the infiltration into the liver (Table 2) nor activation of macrophages (Fig. 3B). Endotoxin administration increased serum TNFa concentration 1 hour later in P. acnes–pretreated rats. Such increase was not attenuated by treatment with both anti–ICAM-1 and LFA-1a antibodies (Table 2).
Formazan deposition in hepatic macrophages was not so different among three rats in both the control and gum arabic–treated groups. Hepatic macrophages were prominently activated 7 days after P. acnes administration, because marked formazan deposition was observed in macrophages throughout the liver in the control group (Fig. 3C). Treatment with gum arabic attenuated such activation of macrophages (Fig. 3D). As shown in Table 4, the control group demonstrated the increased serum ALT values 5 hours after endotoxin administration, and this increase was significantly attenuated by treatment with gum arabic. On liver histology, all of the control group showed coagulative necrosis of grade 3 in the midzonal areas. In contrast, two of six rats showed coagulative necrosis of grade 1, and remaining rats the necrosis of grade 2 in the gum arabic–treated group.
Effect of Rabbit Antisera Against Polymorphonuclear Leukocytes on Peripheral Neutrophils and Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment IV)
DISCUSSION
As shown in Table 3, administration of antisera against polymorphonuclear leukocytes significantly reduced the count of peripheral neutrophils to 0.89% of that in the control group. The control group showed the increased serum ALT values and prolonged prothrombin time 5 hours after endotoxin administration.
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To elucidate the reason why activated macrophages can destroy exclusively endothelial cells in the hepatic sinusoids in massive hepatic necrosis provoked by endotoxin in rats pretreated with P. acnes, the immunohistochemical expression of ICAM-1 and LFA-1a in those cells and the effect of antibodies against both adhesion molecules on this liver injury were investigated. Because circulating cytotoxic neutrophils are reported to express LFA-1a as well,29 and thus, they
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FIG. 3. Light micrographs showing formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate in P. acnes–treated rats. (Original magnification 1132.) (A) Nonspecific IgG-treated group (experiment III). Marked formazan deposition can be seen in macrophages throughout the liver. (B) Antibody - treated group (experiment III). Marked formazan deposition can be observed in macrophages throughout the liver. The extent of the deposition is the same as that in the control group. (C) Saline-treated group (experiment V). Marked formazan deposition can be observed in macrophages throughout the liver. (D) Gum arabic–treated group (experiment V). Faint formazan deposition can be found in macrophages throughout the liver.
might attack endothelial cells if the cells express enough ICAM-1, the effect of antibody against polymorphonuclear leucocytes was also studied. Besides, the role of activated hepatic macrophages was confirmed by blocking macrophage function with an agent other than gadolinium chloride, by which we have already showed the attenuation of this liver injury,19 as gadolinium chloride might act on other cells. As shown in Fig. 1, there were increased stains of
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ICAM-1 and LFA-1a in hepatic sinusoidal cells in P. acnes–treated liver compared with normal liver. Such staining of both adhesion molecules became more prominent 3 hours after endotoxin administration, preceding the development of liver necrosis. From immunohistological findings on electron microscopy, this increase was proved to be caused by the expression in sinusoidal endothelial cells and macrophages, respectively, as shown in Fig. 2. ICAM-1 was not stained in
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TABLE 3. Effect of Antisera Against Polymorphonuclear Leukocytes on Peripheral Neutrophils and the Extent of Liver Injury in P. acnes–Treated Rats 0 and 5 Hours After Endotoxin Administration, Respectively (Experiment IV) Counts of Peripheral Neutrophils (/mm3)
Serum ALT (KU)
Groups
0
5
Control Antisera-treated
902 { 74† 8 { 5§
262 { 141 574 { 406
Extent of Liver Injury* (Grade) Prothrombim Time (s)
0
1
5 (hr after endotoxin administration)
16.7 { 2.3 18.4 { 2.6
0 0
2
3
5 5
1‡ 1
5
0 0
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as shown in the footnote of Table 1. † Values given as mean { SD of six rats. ‡ No. of rats. § P õ .01 vs. control group, by Student’s t test.
hepatocytes in this model, although hepatocytes are reported in vitro to express ICAM-1 in response to cytokines.38,39 Considering that endothelial cell destruction occurs from 1 to 3 hours after endotoxin administration in P. acnes–treated rats,19 hepatic macrophages should adhere to endothelial cells but not to hepatocytes via ICAM-1 and LFA-1a during this period. Such findings seem to be parallel with our previous observation that hepatocytes are not directly destroyed by activated hepatic macrophages in isolated perfused liver system of P. acnes–treated rats,19 despite the fact that the destruction is produced in vitro by activated macrophages.20-22 Treatment of liver injury with monoclonal antibodies against ICAM-1 and LFA-1a was started 3 days before P. acnes administration to provide a sufficient effectiveness for both adhesion molecules in endothelial cells and macrophages expressed markedly 7 days after P. acnes administration. To exclude the possibility that both antibodies affected the infiltration into the liver and the stimulatory stage of hepatic macrophages, the volumes of liver and granuloma and the ability of hepatic macrophages to produce superoxide anions by liver perfusion with NBT and PMA32,33 were determined. Also, TNFa concentrations were measured in
TABLE 4. Effect of Gum Arabic on the Extent of Liver Injury 5 Hours After Endotoxin Administration in P. acnes– Treated Rats (Experiment V) Extent of Liver Injury* (Grade) Groups
Serum ALT (KU)
0
1
2
3
Control Gum arabic–treated
1,942 { 440† 897 { 272§
0 0
0 2
0 4
6‡ 0\
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as shown in the footnote of Table 1. † Values given as mean { SD of six rats. ‡ No. of rats. § P õ .01 vs. control group, by Student’s t test. \ P õ .01 vs. control group, by Mann-Whitney test.
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the sera 1 hour after endotoxin administration, when the concentration increased to the highest level in P. acnes–treated rats according to our preliminary experiments. As shown in Tables 1 and 2, treatment with both antibodies markedly attenuated the liver injury assessed by serum ALT activity, blood coagulation parameters, and liver histology, but did not change the infiltration and stimulatory stage of hepatic macrophages. In contrast, treatment with anti–ICAM-1 or LFA-1a antibody alone was not so effective for the attenuation of liver injury. The reason for this is unclear, but similar observations have been reported in various types of tissue damage.1,5 Differences in adhesion sites and antigen sites on adhesion molecule proteins should be investigated. To investigate the contribution of neutrophils to the liver injury, antiserum against rat polymorphonuclear leukocytes was employed, because the antiserum is shown to agglutinate polymorphonuclear leukocytes but not macrophages or monocytes in vitro (Inter-Cell Technologies, Inc., Unpublished observations, August 1995). As shown in Table 3, the amount of the antiserum used eradicated almost completely circulating neutrophils but did not affect the liver injury. This may imply that neutrophils play a minor role, if any, in the development of this liver injury. As an agent to block hepatic macrophage function, gum arabic was used, because gum arabic is a polysaccharide with molecular weight of approximately 0.5 to 1.4 1 106,40 which has no hepatoprotective action according to our preliminary experiments, and its administration can suppress the activation of hepatic macrophages after partial hepatectomy in rats.34 As shown in Fig. 3D and Table 4, gum arabic administration attenuated the activation of hepatic macrophages after P. acnes administration, the derangement of serum ALT values, and the histological extent of hepatic necrosis after endotoxin injection. Thus, it is concluded that activated macrophages can adhere to endothelial cells through ICAM-1 and LFA-1a in the hepatic sinusoids and lead to massive hepatic necrosis in this model. Because cytotoxic mediators such as superoxide anions and TNFa released from activated hepatic macrophages directly destroy
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sinusoidal endothelial cells in this model,19 adhesion of activated macrophages to endothelial cells through LFA-1a and ICAM-1 seems to increase the concentration of these cytotoxic mediators on the surface of sinusoidal endothelial cells. Jaeschke and Farhood reported that activated Kupffer cells contributed to ischemiareperfusion liver injury during the first few hours of reperfusion,24 and the contribution of neutrophils was seen several hours later.23 It would be possible that activated hepatic macrophages are responsible for the initiation of liver injury or the contribution of those cells differs in the types of liver injury. These matters should be investigated. Therapeutic efficacy with antibodies against adhesion molecules has been reported in organ injuries induced by neutrophils1,2,25,26,41 and cytotoxic T lymphocytes.5 In the liver, Jaeschke et al reported that functional inactivation of neutrophils with Mac-1 monoclonal antibody attenuated rat liver injuries provoked by galactosamine-endotoxin25 and ischemia-reperfusion.26 Also, they demonstrated that administration of anti–ICAM-1 monoclonal antibody significantly attenuated neutrophil-induced liver injury.41 The current report showed the effectiveness of anti–ICAM-1 and LFA-1a antibodies in liver injury induced by hepatic macrophages. Acknowledgment: We thank Dr Mituaki Isobe for useful advice and Kayoko Naiki for technical help. REFERENCES 1. Iigo Y, Takahashi T, Tamatani T, Miyasaka M, Higashida T, Yagita H, Okumura K, et al. ICAM-1 dependent pathway is critically involved in the pathogenesis of adjuvant arthritis in rats. J Immunol 1991;147:4167-4171. 2. Mulligan MS, Varani JV, Dame MK, Lane CL, Smith CW, Anderson DC, Ward PA. Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin Invest 1991;88:1396-1406. 3. Kanagawa K, Ishikura H, Takahashi C, Tamatani T, Miyasaka M, Togashi M, Koyanagi T, et al. Identification of ICAM-1-positive cells in the nongrafted and transplanted rat kidney: an immunohistochemical and ultrastructural study. Transplantation 1991;52:1057-1062. 4. Redl H, Dinges HP, Buurman WA, Van der Linden CJ, Pober JS, Cotran RS, Schlag G. Expression of endothelial leukocyte adhesion molecule-1 in septic but not traumatic/hypovolemic shock in the baboon. Am J Pathol 1991;139:461-466. 5. Isobe M, Yagita H, Okumura K, Ihara A. Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1. Science 1992;255:1125-1127. 6. Volpes R, Van den Oord JJ, Desmet VJ. Immunohistochemical study of adhesion molecules in liver inflammation. HEPATOLOGY 1990;12:59-65. 7. Volpes R, Van den Oord JJ, Desmet VJ. Hepatic expression of intercellular adhesion molecule-1 (ICAM-1) in viral hepatitis B. HEPATOLOGY 1990;12:148-154. 8. Malizia G, Dino O, Pisa R, Caltagirone M, Giannuoli G, Marco VD, Aragona E, et al. Expression of leukocyte adhesion molecule in the liver of patients with chronic hepatitis B virus infection. Gastroenterology 1991;100:749-755. 9. Adams DH, Hubscher SG, Shaw J, Johnson GD, Babbs C, Rothlein R, Neuberger JM. Increased expression of intercellular adhesion molecule-1 on bile duct in primary biliary cirrhosis and primary sclerosing cholangitis. HEPATOLOGY 1991;14:426-431. 10. Volpes R, Van den Oord JJ, Desmet VJ. Vascular adhesion molecule in acute and chronic liver inflammation. HEPATOLOGY 1992;15:269-275.
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11. Hubscher PB, Shaw J, Elias E, Adams DH. Is the intercellular adhesion molecule-1/leukocyte function-associated antigen 1 pathway of leukocyte adhesion involved in the tissue damage of alcoholic hapatitis? Gut 1992;33:268-271. 12. Fujiwara K, Ogata I, Ohta Y, Hirata K, Oka Y, Yamada S, Sato Y, et al. Intravascular coagulation in acute liver failure in rats and its treatment with antithrombin III. Gut 1988;29:11031108. 13. Hirata K, Ogata I, Ohta Y, Fujiwara K. Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats. J Pathol 1989;158:157-165. 14. Ferluga J, Allison AC. Role of mononuclear infiltrating cells in pathogenesis of hepatitis. Lancet 1978;ii:610-611. 15. Arthur MJP, Bentley IS, Tanner AR, Kowalski-Saunders P, Wright R. Oxygen-derived free radicals promote hepatic injury in the rat. Gastroenterology 1985;89:1114-1122. 16. Arthur MJP, Kowalski-Saunders P, Wright R. Corynebacterium parvum-elicited hepatic macrophages demonstrate enhanced respiratory burst activity compared with resident Kupffer cells in the rat. Gastroenterology 1986;91:174-181. 17. Yamada S, Ogata I, Hirata K, Mochida S, Tomiya T, Fujiwara K. Intravascular coagulation in the development of massive hepatic necrosis induced by Corynebacterium parvum and endotoxin in rats. Scand J Gastroenterol 1989;24:293-298. 18. Nagakawa J, Hishinuma I, Hirota K, Miyamoto K, Yamanaka T, Tsukidate K, Katayama K, et al. Involvement of tumor necrosis factor-a in the pathogenesis of activated macrophage-mediated hepatitis in mice. Gastroenterology 1990;99:758-765. 19. Arai M, Mochida S, Ohno A, Ogata I, Fujiwara K. Sinusoidal endothelial cell damage by activated hepatic macrophages in rat liver necrosis. Gastroenterology 1993;104:1466-1471. 20. Mizoguchi Y, Tsutsui H, Miyajima K, Sakagami Y, Seki S, Kobayashi K, Yamamoto S, et al. The protective effects of prostaglandin E1 in an experimental massive hepatic cell necrosis model. HEPATOLOGY 1987;7:1184-1188. 21. Shiratori Y, Kawase T, Shiina S, Okano K, Sugimoto T, Teraoka H, Matano S, et al. Modulation of hepatotoxicity by macrophages in the liver. HEPATOLOGY 1988;8:815-821. 22. Funatsu K, Matsumaru A, Ueno M, Mizuno Y, Nishida J, Oda M, Tsuchiya M. Electron microscopic and biochemical study of hepatocellular injury by activated macrophages. J Clin Electron Microsc 1989;22:5-6. 23. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J 1990;4:3355-3359. 24. Jaeschke H, Farhood A. Neutrophils and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am J Physiol 1991;260:G355-G356. 25. Jaeschke H, Farhood A, Smith CW. Neutrophils-induced liver cell injury in endotoxin shock is a CD11b/CD18-dependent mechanisms. Am J Physiol 1991;261:G1051-G1056. 26. Jaeschke H, Farhood A, Bautista AP, Spolarics Z, Spitzer JJ, Smith CW. Functional inactivation of neutrophils with Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemiareperfusion injury in rat liver. HEPATOLOGY 1993;17:915-932. 27. Hynes RO. Integlins: a family of cell surface receptors. Cell 1987;108:549-554. 28. Dustin ML, Springer TA. Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J Cell Biol 1988;107:321331. 29. Larson RS, Springer TA. Structure and function of leukocyte integrins. Immunol Rev 1990;114:181-217. 30. Tamatani T, Miyasaka M. Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells. Int Immunol 1990;2:165-171. 31. Tamatani T, Kotani M, Miyasaka M. Characterization of the rat leukocyte integrin, CD11/CD18, by use of LFA-1 subunit-specific monoclonal antibodies. Eur J Immunol 1991;21:627-633. 32. Ogata I, Mochida S, Fujiwara K. Formazan formation in hepatic
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33.
34. 35.
36.
37.
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macrophages after nitro blue tetrazolium perfusion into rat liver. Biomed Res 1988;9:113-117. Mochida S, Ogata I, Ohta Y, Yamada S, Fujiwara K. In situ evaluation of the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions in rats. J Pathol 1989;158:67-71. Mochida S, Ogata I, Hirata K, Ohta Y, Yamada S, Fujiwara K. Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats. Gastroenterology 1990;99:771-777. Fujiwara K, Ogata I, Mochida S, Yamada S, Hirata K, Tomiya T, Ohta Y. Activated Kupffer cells as a factor of massive hepatic necrosis after liver resection. Hepatogastroenterology 1990;37: 194-197. Yamada S, Mochida S, Ohno A, Hirata K, Ogata I, Ohta Y, Fujiwara K. Evidence for enhanced secretory function of hepatic macrophages after long-term ethanol feeding in rats. Liver 1991;11:220-224. Mochida S, Ohta Y, Ogata I, Fujiwara K. Gut-derived substances
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38.
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in activation of hepatic macrophages after partial hepatectomy in rats. J Hepatol 1992;16:266-272. Volpes R, Van den Oord JJ, Desmet VJ, Yap SH. Induction of intercellular adhesion molecule-1 (CD54) on human hepatoma cell line HepG2: influence of cytokines and hepatitis B virusDNA transfection. Clin Exp Immunol 1992;87:71-75. Kvale D, Brandtzaeg P. Immune modulation of adhesion molecule ICAM-1 (CD54) and LFA-3 (CD58) in human hepatocyte cell line. J Hepatol 1993;17:347-352. Maclean Ross AH, Eastwood MA, Brydon WG, Anderson JR, Anderson DMW. A study of the effects dietary gum arabic in humans. Am J Clin Nutr 1983;37:368-375. Essani N, Fisher MA, Farhood A, Manning AM, Smith CW, Jaeschke H. Cytokine-induced upregulation of hepatic intercellular adhesion molecule-1 messenger RNA expression and its role in the pathophysiology of murine endotoxin shock and acute liver failure. HEPATOLOGY 1995;21:1632-1639.
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Massive hepatic necrosis develops after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes as a result of microcirculatory disturbance caused by endothelial cell destruction by activated macrophages in the hepatic sinusoids. Immunohistochemical hepatic expression of intercellular adhesion molecule–1 (ICAM-1) and lymphocyte function– associated antigen 1a (LFA-1a) and the effect of monoclonal antibodies against both adhesion molecules on liver necrosis provoked after endotoxin administration was studied in these rats. There were increased stains of ICAM-1 in endothelial cells and LFA-1a in macrophages in the hepatic sinusoids in Propionibacterium acnes–pretreated rats compared with normal rats. Such stains were further increased soon after endotoxin administration, followed by development of hepatic necrosis. Monoclonal antibodies against both adhesion molecules significantly attenuated the extent of liver injury compared with controls, without affecting the infiltration and activation of hepatic macrophages. Polyclonal antibodies against polymorphonuclear leukocytes eradicated circulating neutrophils, but did not change such liver injury, although gum arabic, which suppressed macrophage activation, attenuated the extent of liver injury. Thus, adhesion between endothelial cells and activated macrophages in the hepatic sinusoids via ICAM1 and LFA-1a is essential for the initiation of massive hepatic necrosis of this type. Contribution of neutrophils seems less likely. (HEPATOLOGY 1996;23:320-328.)
Cell-to-cell interaction through adhesion molecules has attracted attention in the pathogenesis of various organ injuries.1-5 Treatments using antibodies against intercellular adhesion molecule 1 (ICAM-1) and lym-
Abbreviations: ICAM-1, intercellular adhesion molecule 1; LFA-1a, lymphocyte function–associated antigen 1a; PBS, phosphate-buffered saline; Ig, immunoglobulin; NBT, nitro blue tetrazolium; PMA, phorbol 12-myristate 13acetate; TNF, tumor necrosis factor; ALT, alanine transaminase. From the 1Third Department of Internal Medicine, Saitama Medical School, Saitama, Japan; 2First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan; 3Japan Tabacco Inc., Pharmaceutical Basic Research Center, Kanagawa, Japan; and 4Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Osaka, Japan. Received March 8, 1995; accepted September 7, 1995. Address reprint requests to: Kenji Fujiwara, M.D., Ph.D., Third Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyamacho, Iruma-gun, Saitama 350-04, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2302-0018$3.00/0
phocyte function–associated antigen 1a (LFA-1a) demonstrated the contribution of these adhesion molecules to induction of arthritis by adjuvant in rats1 and acute rejection after cardiac transplantation in mice.5 Adhesion molecules also seem to play a role in the development of liver injury, as the expression of several adhesion molecules in human liver has been reported to increase in association with liver diseases.6-11 Our previous observations demonstrate that fibrin deposition due to endothelial cell destruction in the hepatic sinusoids causes massive hepatic necrosis,12,13 and such endothelial cell destruction can be produced by activated hepatic macrophages in massive hepatic necrosis induced by endotoxin in rats pretreated with heat-killed Propionibacterium acnes (P. acnes; Corynebacterium parvum).14-19 With isolated perfused liver system of this model, activated hepatic macrophages are proven to destroy sinusoidal endothelial cells but not hepatocytes.19 However, the reason why activated macrophages exclusively attack endothelial cells in the hepatic sinusoids despite the fact that macrophages can damage hepatocytes in vitro20-22 is to be elucidated. Also, the contribution of other cells to massive hepatic necrosis is important, because cytotoxic cells such as neutrophils are circulating in vivo.1,2,23-26 ICAM-1 and its ligand, LFA-1a, have been reported to be expressed in isolated endothelial cells and macrophages, respectively.27,28 This prompted us to investigate the role of both adhesion molecules in the interaction between endothelial cells and activated macrophages in the hepatic sinusoids in massive hepatic necrosis of this type and the contribution of neutrophils as they are shown to express LFA-1a as well.29 MATERIALS AND METHODS Experimental Designs Male Fisher rats (Charles River Japan, Atugi, Japan) weighing 170 to 190 g were maintained on a commercial pelleted diet and water ad libitum in a room at 22 { 27C under normal laboratory lighting conditions. All animal study protocols conformed to National Research Council criteria for humane care. Experiment I. Eighteen rats received 15 mg/kg body weight heat-killed P. acnes (C. parvum whole cells: Ribi Immunochem Research Inc., Hamilton, MO) as a 7.5 mg/mL suspension in saline through the femoral vein. On the 7th day, they were given an intravenous injection of 200 mg/kg endotoxin (lipopolysaccharide from Escherichia coli 026:B6;
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Difco, Detroit, MI) as a 100 mg/mL solution in saline, and were killed 0, 3, or 5 hours later for immunohistochemical examination of the liver on light or electron microscopy. Six normal rats were also killed for a similar examination. Experiment II. Eighteen rats received a daily intraperitoneal injection of 1 mg/kg body weight of anti-rat ICAM-1 and/ or LFA-1a monoclonal antibodies as 1 mg/3 mL solutions in phosphate-buffered saline (PBS) for 10 days (ICAM-1 and LFA-1–, and ICAM-1–, and LFA-1–treated groups). Another 12 rats were given similarly 1 or 2 mg/kg body weight of nonimmune mice immunoglobulin (Ig)G as a 1 mg/3 mL solution in PBS (control group). All rats received heat-killed P. acnes on the 4th day and endotoxin 24 hours after the final antibody treatment. For the determination of the extent of liver injury, blood was collected through the inferior caval vein under anesthesia with ether 5 hours after endotoxin injection, with an empty plastic syringe and a plastic syringe containing 1:10 volume of 3.8% sodium citrate solution to prepare serum and plasma, respectively, and the liver was excised. Experiment III. Twenty rats received both antibodies or nonimmune mice IgG and heat-killed P. acnes similarly. Eight rats were subjected to liver perfusion with nitro blue tetrazolium (NBT) and phorbol 12-myristate 13-acetate (PMA) 24 hours after the final antibody treatment for the evaluation of the activation and infiltration of hepatic macrophages. Another 12 rats received an intravenous injection of endotoxin 24 hours after the final antibody treatment, and 1 hour later blood was collected through the inferior caval vein under anesthesia with ether for the determination of serum tumor necrosis factor-a (TNFa) concentration. Experiment IV. Twelve rats received heat-killed P. acnes similarly. On the 6th day, They were given an intravenous injection of 0.2 mL/kg rabbit antisera against rat polymorphonuclear leukocytes (Inter-Cell Technologies, Inc., Somerville, NJ) (antisera-treated group) or the same volume of normal rabbit sera (control group) as 10% solution in saline. Twenty-four hours later, 0.6 mL blood was collected through the femoral vein using a plastic syringe containing 60 units of heparin (Novo Industri A/S, Copenhagen, Denmark) for the count of peripheral neutrophils, and then the rats were given an intravenous injection of endotoxin similarly. Five hours after endotoxin administration, blood was collected through the inferior vena cava for the measurement of the parameters of liver injury under anesthesia with ether, and the liver was excised for the histological examination. Experiment V. Eighteen rats received a daily intraperitoneal injection of 100 mg gum arabic (Sigma Chemical Company, St. Louis, MO) as 10% solution in saline (gum arabic– treated group) or the same volume of saline (control group) for 10 days. Six rats in both groups were given heat-killed P. acnes and endotoxin on 4th and 11th days, respectively. Five hours after endotoxin administration, blood was collected through the inferior vena cava for the determination of the extent of liver injury under anesthesia with ether, and the liver was excised for the histological examination. Another three rats in both groups were given heat-killed P. acnes on the 4th day, and they were subjected to liver perfusion with NBT and PMA on the 11th day for the evaluation of hepatic macrophage activation. Preparation of Anti-rat ICAM-1, LFA-1a Monoclonal Antibodies and Nonimmune Mice IgG Monoclonal antibodies used were anti-rat ICAM-1, 1A29 (mouse IgG1)30 and anti-rat LFA-1a, WT1 (mouse IgG2a).31 Hybridoma cells that produce both monoclonal antibodies
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were cultured in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 10% fetal bovine serum (Gibco Laboratories, Life Technologies Inc., Grand Island, NY) for several days. Then cultured hybridoma cells were injected into the abdominal cavity of female Balb/c nude mice (Charles River Japan) pretreated with 0.5 mL of 2,6,10,14-tetramethyl-pentadecane intraperitoneally, and ascites were collected 7 days later. Both antibodies were purified from the ascites using protein G affinity column (Protein G Sepharose 4 Fast Flow; Pharmacia, Uppsala, Sweden), and concentrated using ultrafiltration membrane (AMICON; W.R. Grace & Co., Danver, MA).5 Control IgG was purified similarly from nonimmune mice serum (Chemicon International Inc., Temecula, CA). Immunohistochemical Study of the Liver Under anesthesia with an intraperitoneal injection of 50 mg/kg pentobarbital sodium, the abdomen was cut open. For light microscopic examination, the liver was perfused by the portal vein with saline at a flow rate of 10 mL/minute for 1 minute, and excised. Blocks of 5 mm3 cut off with a blade from excised livers were immersed into ornithine-carbamoyltransferase Compound (Miles Inc., Elkhart, IN), snap-frozen in liquid nitrogen, and stored at 0807C. From frozen blocks, 5-mm cryostat sections were made and fixed in absolute acetone for 10 minutes. Endogenous peroxidase activity was blocked in methanol containing 0.3% hydrogen peroxide for 15 minutes, and nonspecific binding sites in whole rabbit serum for 20 minutes. Then a two-step indirect immunoperoxidase procedure was performed; sections were incubated with anti-rat ICAM-1 or LFA-1a monoclonal antibody as a solution of 1 mg/mL in PBS for 1 hour, and then with peroxidase-conjugated rabbit anti-mouse IgG (working dilution 1:100, Vector Laboratories, Inc., Burlingame, CA) for 1 hour at room temperature. The reaction product was developed by incubation for 5 minutes in solution of 0.05% 3,3*-diaminobenzidine, pH 7.2, containing 0.01% hydrogen peroxide (Vector Laboratories). Counterstaining was done in 4% methyl green solution for 2 minutes. In controls, nonimmune mouse IgG was used instead of primary antibodies. For electron microscopic examination, the liver was perfused with saline in the same manner as for light microscopic examination, and with cold fixative containing 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mol/L PBS. Three blocks of 5 mm3 cut off with a blade from the excised liver were immersed and shaken in PBS containing 5% sucrose, and successively in PBS containing 10%, 20%, 30%, and 40% sucrose at 47C. The blocks were then put into ornithine-carbamoyltransferase (OCT) compound and rapidly frozen in dry ice–acetone. Eight-micrometer cryostat sections were made from frozen blocks. A two-step indirect immunoperoxidase procedure was performed in the same way as for light microscopic examination. After fixation of the sections in 2.5% glutaraldehyde solution, the reaction product was developed by incubation in a 0.05% dimethylaminoazobenzene solution without H2O2 and then in dimethylaminoazobenzene solution containing 0.005% H2O2 . The sections were postfixed in 1% osmium tetroxide and dehydrated through graded concentrations of ethanol. Epoxy resin in gelatin capsules was placed on the section at 607C. Ultrathin sections made with a glass knife were observed under Hitachi (Hitachi, Tokyo, Japan) H-800 electron microscope at 100 kV. Procedure for Liver Perfusion With Nitro Blue Tetrazolium and Phorbol Myristate Acetate As previously described,32,33 after injection of 200 units of heparin into the inferior vena cava under anesthesia with an
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TABLE 1. Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Liver Function Tests and Histological Extent of Liver Injury 5 Hours After Endotoxin Administration in P. acnes–Pretreated Rats (Experiment II)
Groups
Control (IgG: 2 mg/kg) ICAM-1– and LFA-1a –treated Control (IgG: 1 mg/kg) ICAM-1–treated LFA-1a –treated
Serum ALT (KU)
450.8 175.0 662.9 666.7 326.7
{ { { { {
196.7† 110.0§ 380.5 260.7 220.4
Prothrombin Time (s)
Plasma Antithrombin III (%)
0
1
2
3
22.7 15.6 22.8 20.8 21.2
60.2 75.2 52.3 49.2 48.3
0 1 0 0 0
1 4 0 0 0
1 1 0 0 1
4‡ 0Ø 6 6 5
{ { { { {
4.5 2.2\ 4.7 3.0 4.8
{ { { { {
10.3 8.9§ 6.4 5.2 11.2
Extent of Liver Injury* (Grade)
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as follows: grade 0, no coagulative necrosis; grade 1, coagulative necrosis covering õ5%; grade 2, 5% to 10%; grade 3, ú10%. † Values given as mean { SD of six rats. Normal values of serum ALT, prothrombin time, and plasma antithrombin III are 22 { 6 KU, 10.1 { 0.6 seconds, and 105.0% { 2.7%, respectively (n Å 4). ‡ No. of rats. § P õ .05. \ P õ .01 vs. control (IgG: 2 mg/kg) group, by Student’s t test. Ø P õ .05 vs. control (IgG: 2 mg/kg) group, by Mann-Whitney test.
intraperitoneal injection of pentobarbital sodium, the liver was perfused successively with Ca2/- and Mg2/-free Hanks’ balanced salt solution (Gibco Laboratories) for 5 minutes, Dulbecco’s minimal essential medium (Nissui Pharmaceutical) with 0.5 mg/mL PMA (Sigma Chemical Company) for 5 minutes, 0.05% NBT (Grade III, Sigma Chemical Company) in Eagle’s minimal essential medium with PMA for 10 minutes, and finally with Hanks’ balanced salt solution for 5 minutes. The perfusion was performed at a flow rate of 20 mL/min at 377C with continuous O2 bubbling. All the perfusates contained 20 mmol/L HEPES, pH 7.4. Then the excised liver was fixed in formalin and embedded in paraffin. Evaluation of the Extent of Liver Injury The extent of liver injury was assessed from serum alanine transaminase (ALT) activity, plasma antithrombin III activity, and prothrombin time as well as from the histological extent of liver necrosis. Serum ALT activity was determined using a commercial kit (Iatron Laboratories Inc., Tokyo, Japan). Prothrombin time was measured in plasma using SIMPLASTIN (Division of Warner Lambert Co., Cockeysville, NJ) and a fibrometer (Division of Beckton, Dickinson and Company, Cockeysville, MD). Plasma antithrombin III concentration was determined with Coatest Antithrombin III kit (Kabi Diagnostica, Stockholm, Sweden). Histological examination was performed using formalinfixed and paraffin-embedded liver specimens. All slides were evaluated at random by one of the authors on light microscopy. The extent of liver necrosis was classified using a four-
degree score as shown in the footnote of Table 1 with sections counterstained with hematoxylin-eosin. Evaluation of the Infiltration and Activation of Hepatic Macrophages The activation of hepatic macrophages was based on their stimulatory stage determined by the extent of formazan deposition on sections of the liver after perfusion with NBT and PMA, which were counterstained with Kernechtrot as previously reported.32-37 The infiltration of macrophages into the liver was estimated from liver weights and the size and number of granulomas on the same sections of the liver after perfusion with NBT and PMA counterstained with Masson’s trichrome, as detailed in the footnote of Table 2. All slides were evaluated at random by one of the authors on light microscopy. The activation and infiltration of hepatic macrophages were also estimated from serum concentration of TNFa using a commercial enzyme-linked immunosorbent assay kit (Factor Test Mouse TNF-a kit: Genzyme Diagnostics, Boston, MT). Count of Peripheral Neutrophils The number of total peripheral white blood cells was counted by light microscopy using a Neubauer hemocytemeter after diluting the heparinized blood to 1:10 in volume in Turk’s fluid. Peripheral neutrophil count was calculated from this number and the relative number of neutrophils to white blood cells, which was determined on blood smear stained by Wright and Giemsa.
TABLE 2. Effect of Anti-Rat ICAM-1 and LFA-1a on Infiltration and Activation of Hepatic Macrophages 7 Days After P. acnes Administration in Rats (Experiment III) Granuloma* Groups
Liver Weight (% Body Weight)
No.
Size (mm)
Serum TNFa Concentration (pg/mL)
Control ICAM-1– and LFA-1a –treated
5.73 { 0.33† 5.63 { 0.40
115.0 { 22.7 118.3 { 23.2
57.4 { 5.2 64.5 { 5.0
1,097 { 407‡ 1,463 { 454
* The number of granulomas is total numbers counted from five fields randomly selected under magnification at 100X. The size is the mean of largest diameter of 30 granulomas randomly selected under magnification at 200X. † Values given as mean { SD of four rats. Normal liver weight is 4.26% { 0.45% of body weight (n Å 4). ‡ Values given as mean { SD of six rats.
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FIG. 1. Light micrographs showing immunohistochemical expression of ICAM-1 and LFA-1a in the liver in normal and Propionibacterium acnes (P. acnes)–treated rats (experiment I). (A) ICAM-1 staining in normal liver. (Original magnification 1204.) Very slight stain is seen in sinusoidal cells throughout the liver. (B) LFA-1a staining in normal liver. (Original magnification 1270.) Faint stain is observed in sinusoidal cells in the periportal area (arrows). (C) ICAM-1 staining in P. acnes–treated liver. (Original magnification 1326.) Moderate stain is seen in sinusoidal cells throughout the liver, and marked stain in granulomas (arrowhead). (D) LFA-1a staining in P. acnes–treated liver. (Original magnification 1270.) Faint stain is observed in sinusoidal cells throughout the liver (arrows), and marked stain in granulomas. (E) ICAM-1 staining in the liver 3 hours after endotoxin treatment after pretreatment with P. acnes. (Original magnification 1326.) Marked stain is seen in sinusoidal cells and granulomas (arrowhead) throughout the liver, but not in hepatocytes. (F) LFA-1a staining in the liver 3 hours after endotoxin treatment after pretreatment with P. acnes. (Original magnification 1326.) Marked stain is seen in sinusoidal cells (arrows) and granulomas (arrowhead) throughout the liver.
RESULTS Immunohistochemical Expression of ICAM-1 and LFA-1a in Rat Livers (Experiment I)
Immunohistochemical findings of ICAM-1 and LFA1a in the liver were not so different among three rats in each group. Controls of these adhesion molecules showed no staining. In normal liver, sinusoidal cells showed slight ICAM1 stains throughout the liver and LFA-1a stains in the periportal area on light microscopy (Fig. 1A and B). Electron microscopic examination demonstrated that sinusoidal cells with such slight ICAM-1 and LFA-1a staining were endothelial cells and Kupffer cells, respectively. P. acnes–treated rats showed scattered granulomas and infiltrating macrophages in the hepatic sinusoids on the 7th day and stains of ICAM-1 and LFA-1a in sinusoidal cells throughout the liver more prominently compared with normal rats (Fig. 1C and D). Such stains of both adhesion molecules were further increased 3 hours after endotoxin administration, but hepatic necrosis was not present. Both adhesion molecules were also stained in granulomas, but not in hepatocytes (Fig. 1E and F ). Electron-microscopic examination showed that sinusoidal cells with such
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marked ICAM-1 and LFA-1a staining were endothelial cells and macrophages, respectively (Fig. 2A and B). Coagulative necrosis was observed in the midzonal areas 5 hours after endotoxin administration. In these rats, marked stains of ICAM-1 and LFA-1a were found in sinusoidal endothelial cells and hepatic macrophages, respectively, throughout nonnecrotic areas. Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment II)
As shown in Table 1, endotoxin administration produced increased serum ALT activity, prolonged prothrombin time and decreased plasma AT III concentration 5 hours later in P. acnes–pretreated rats. These derangements were significantly attenuated by treatment with both anti–ICAM-1 and LFA-1a antibodies. Such derangements, however, were not attenuated by the treatment with anti–ICAM-1 or LFA-1a antibody alone. On liver histology, all rats showed coagulative necrosis in the midzonal areas in the control groups; four of six rats and all of six rats showed the necrosis of grade 3 in 2 mg/kg and 1 mg/kg control groups, respectively.
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FIG. 2. Electron micrographs showing immunohistochemical expression of ICAM-1 and LFA-1a in the liver 3 hours after endotoxin administration in P. acnes–treated rats (experiment I). (A) ICAM-1. Sinusoidal endothelial cells show ICAM-1 stains along with the membrane of the cells. There is no stain in hepatocytes. (Original magnification 111,200.) (B) LFA-1a. Staining can be seen on the membrane of hepatic macrophage. Hepatocyte and endothelial cells show no stain. (Original magnification 113,800.)
In contrast, five of six rats showed the necrosis of grade 0 or 1 in the treatment group with both antibodies. However, treatment with anti–ICAM-1 or LFA-1a antibody alone did not affect the histological extent of liver injuries.
On liver histology, all rats showed coagulative necrosis of grade 2 or 3 in the midzonal areas. Administration of the antisera affected neither the deranged liver function tests nor the histological extent of liver injury in P. acnes– and endotoxin-treated rats.
Effect of Anti-Rat ICAM-1 and LFA-1a Antibodies on Infiltration and Activation of Hepatic Macrophages in P. acnes–Treated Rats (Experiment III)
Effect of Gum Arabic on Activation of Hepatic Macrophages and Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment V)
Formazan deposition in hepatic macrophages was not so different among three rats in both control and antibody-treated groups. As shown in Table 2, infiltration of macrophages into the liver was remarkable 7 days after P. acnes administration in the control group, because liver weight and the number and size of hepatic granulomas were significantly increased compared with normal rats. Those macrophages were prominently activated, because they contained marked formazan deposition (Fig. 3A). Treatment with both antibodies affected neither the infiltration into the liver (Table 2) nor activation of macrophages (Fig. 3B). Endotoxin administration increased serum TNFa concentration 1 hour later in P. acnes–pretreated rats. Such increase was not attenuated by treatment with both anti–ICAM-1 and LFA-1a antibodies (Table 2).
Formazan deposition in hepatic macrophages was not so different among three rats in both the control and gum arabic–treated groups. Hepatic macrophages were prominently activated 7 days after P. acnes administration, because marked formazan deposition was observed in macrophages throughout the liver in the control group (Fig. 3C). Treatment with gum arabic attenuated such activation of macrophages (Fig. 3D). As shown in Table 4, the control group demonstrated the increased serum ALT values 5 hours after endotoxin administration, and this increase was significantly attenuated by treatment with gum arabic. On liver histology, all of the control group showed coagulative necrosis of grade 3 in the midzonal areas. In contrast, two of six rats showed coagulative necrosis of grade 1, and remaining rats the necrosis of grade 2 in the gum arabic–treated group.
Effect of Rabbit Antisera Against Polymorphonuclear Leukocytes on Peripheral Neutrophils and Liver Injury Induced by Endotoxin After P. acnes Administration in Rats (Experiment IV)
DISCUSSION
As shown in Table 3, administration of antisera against polymorphonuclear leukocytes significantly reduced the count of peripheral neutrophils to 0.89% of that in the control group. The control group showed the increased serum ALT values and prolonged prothrombin time 5 hours after endotoxin administration.
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To elucidate the reason why activated macrophages can destroy exclusively endothelial cells in the hepatic sinusoids in massive hepatic necrosis provoked by endotoxin in rats pretreated with P. acnes, the immunohistochemical expression of ICAM-1 and LFA-1a in those cells and the effect of antibodies against both adhesion molecules on this liver injury were investigated. Because circulating cytotoxic neutrophils are reported to express LFA-1a as well,29 and thus, they
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FIG. 3. Light micrographs showing formazan deposition in hepatic macrophages after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate in P. acnes–treated rats. (Original magnification 1132.) (A) Nonspecific IgG-treated group (experiment III). Marked formazan deposition can be seen in macrophages throughout the liver. (B) Antibody - treated group (experiment III). Marked formazan deposition can be observed in macrophages throughout the liver. The extent of the deposition is the same as that in the control group. (C) Saline-treated group (experiment V). Marked formazan deposition can be observed in macrophages throughout the liver. (D) Gum arabic–treated group (experiment V). Faint formazan deposition can be found in macrophages throughout the liver.
might attack endothelial cells if the cells express enough ICAM-1, the effect of antibody against polymorphonuclear leucocytes was also studied. Besides, the role of activated hepatic macrophages was confirmed by blocking macrophage function with an agent other than gadolinium chloride, by which we have already showed the attenuation of this liver injury,19 as gadolinium chloride might act on other cells. As shown in Fig. 1, there were increased stains of
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ICAM-1 and LFA-1a in hepatic sinusoidal cells in P. acnes–treated liver compared with normal liver. Such staining of both adhesion molecules became more prominent 3 hours after endotoxin administration, preceding the development of liver necrosis. From immunohistological findings on electron microscopy, this increase was proved to be caused by the expression in sinusoidal endothelial cells and macrophages, respectively, as shown in Fig. 2. ICAM-1 was not stained in
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TABLE 3. Effect of Antisera Against Polymorphonuclear Leukocytes on Peripheral Neutrophils and the Extent of Liver Injury in P. acnes–Treated Rats 0 and 5 Hours After Endotoxin Administration, Respectively (Experiment IV) Counts of Peripheral Neutrophils (/mm3)
Serum ALT (KU)
Groups
0
5
Control Antisera-treated
902 { 74† 8 { 5§
262 { 141 574 { 406
Extent of Liver Injury* (Grade) Prothrombim Time (s)
0
1
5 (hr after endotoxin administration)
16.7 { 2.3 18.4 { 2.6
0 0
2
3
5 5
1‡ 1
5
0 0
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as shown in the footnote of Table 1. † Values given as mean { SD of six rats. ‡ No. of rats. § P õ .01 vs. control group, by Student’s t test.
hepatocytes in this model, although hepatocytes are reported in vitro to express ICAM-1 in response to cytokines.38,39 Considering that endothelial cell destruction occurs from 1 to 3 hours after endotoxin administration in P. acnes–treated rats,19 hepatic macrophages should adhere to endothelial cells but not to hepatocytes via ICAM-1 and LFA-1a during this period. Such findings seem to be parallel with our previous observation that hepatocytes are not directly destroyed by activated hepatic macrophages in isolated perfused liver system of P. acnes–treated rats,19 despite the fact that the destruction is produced in vitro by activated macrophages.20-22 Treatment of liver injury with monoclonal antibodies against ICAM-1 and LFA-1a was started 3 days before P. acnes administration to provide a sufficient effectiveness for both adhesion molecules in endothelial cells and macrophages expressed markedly 7 days after P. acnes administration. To exclude the possibility that both antibodies affected the infiltration into the liver and the stimulatory stage of hepatic macrophages, the volumes of liver and granuloma and the ability of hepatic macrophages to produce superoxide anions by liver perfusion with NBT and PMA32,33 were determined. Also, TNFa concentrations were measured in
TABLE 4. Effect of Gum Arabic on the Extent of Liver Injury 5 Hours After Endotoxin Administration in P. acnes– Treated Rats (Experiment V) Extent of Liver Injury* (Grade) Groups
Serum ALT (KU)
0
1
2
3
Control Gum arabic–treated
1,942 { 440† 897 { 272§
0 0
0 2
0 4
6‡ 0\
* Histological extent of liver injury is classified according to the area of coagulative necrosis in hepatic lobules as shown in the footnote of Table 1. † Values given as mean { SD of six rats. ‡ No. of rats. § P õ .01 vs. control group, by Student’s t test. \ P õ .01 vs. control group, by Mann-Whitney test.
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the sera 1 hour after endotoxin administration, when the concentration increased to the highest level in P. acnes–treated rats according to our preliminary experiments. As shown in Tables 1 and 2, treatment with both antibodies markedly attenuated the liver injury assessed by serum ALT activity, blood coagulation parameters, and liver histology, but did not change the infiltration and stimulatory stage of hepatic macrophages. In contrast, treatment with anti–ICAM-1 or LFA-1a antibody alone was not so effective for the attenuation of liver injury. The reason for this is unclear, but similar observations have been reported in various types of tissue damage.1,5 Differences in adhesion sites and antigen sites on adhesion molecule proteins should be investigated. To investigate the contribution of neutrophils to the liver injury, antiserum against rat polymorphonuclear leukocytes was employed, because the antiserum is shown to agglutinate polymorphonuclear leukocytes but not macrophages or monocytes in vitro (Inter-Cell Technologies, Inc., Unpublished observations, August 1995). As shown in Table 3, the amount of the antiserum used eradicated almost completely circulating neutrophils but did not affect the liver injury. This may imply that neutrophils play a minor role, if any, in the development of this liver injury. As an agent to block hepatic macrophage function, gum arabic was used, because gum arabic is a polysaccharide with molecular weight of approximately 0.5 to 1.4 1 106,40 which has no hepatoprotective action according to our preliminary experiments, and its administration can suppress the activation of hepatic macrophages after partial hepatectomy in rats.34 As shown in Fig. 3D and Table 4, gum arabic administration attenuated the activation of hepatic macrophages after P. acnes administration, the derangement of serum ALT values, and the histological extent of hepatic necrosis after endotoxin injection. Thus, it is concluded that activated macrophages can adhere to endothelial cells through ICAM-1 and LFA-1a in the hepatic sinusoids and lead to massive hepatic necrosis in this model. Because cytotoxic mediators such as superoxide anions and TNFa released from activated hepatic macrophages directly destroy
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sinusoidal endothelial cells in this model,19 adhesion of activated macrophages to endothelial cells through LFA-1a and ICAM-1 seems to increase the concentration of these cytotoxic mediators on the surface of sinusoidal endothelial cells. Jaeschke and Farhood reported that activated Kupffer cells contributed to ischemiareperfusion liver injury during the first few hours of reperfusion,24 and the contribution of neutrophils was seen several hours later.23 It would be possible that activated hepatic macrophages are responsible for the initiation of liver injury or the contribution of those cells differs in the types of liver injury. These matters should be investigated. Therapeutic efficacy with antibodies against adhesion molecules has been reported in organ injuries induced by neutrophils1,2,25,26,41 and cytotoxic T lymphocytes.5 In the liver, Jaeschke et al reported that functional inactivation of neutrophils with Mac-1 monoclonal antibody attenuated rat liver injuries provoked by galactosamine-endotoxin25 and ischemia-reperfusion.26 Also, they demonstrated that administration of anti–ICAM-1 monoclonal antibody significantly attenuated neutrophil-induced liver injury.41 The current report showed the effectiveness of anti–ICAM-1 and LFA-1a antibodies in liver injury induced by hepatic macrophages. Acknowledgment: We thank Dr Mituaki Isobe for useful advice and Kayoko Naiki for technical help. REFERENCES 1. Iigo Y, Takahashi T, Tamatani T, Miyasaka M, Higashida T, Yagita H, Okumura K, et al. ICAM-1 dependent pathway is critically involved in the pathogenesis of adjuvant arthritis in rats. J Immunol 1991;147:4167-4171. 2. Mulligan MS, Varani JV, Dame MK, Lane CL, Smith CW, Anderson DC, Ward PA. Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J Clin Invest 1991;88:1396-1406. 3. Kanagawa K, Ishikura H, Takahashi C, Tamatani T, Miyasaka M, Togashi M, Koyanagi T, et al. Identification of ICAM-1-positive cells in the nongrafted and transplanted rat kidney: an immunohistochemical and ultrastructural study. Transplantation 1991;52:1057-1062. 4. Redl H, Dinges HP, Buurman WA, Van der Linden CJ, Pober JS, Cotran RS, Schlag G. Expression of endothelial leukocyte adhesion molecule-1 in septic but not traumatic/hypovolemic shock in the baboon. Am J Pathol 1991;139:461-466. 5. Isobe M, Yagita H, Okumura K, Ihara A. Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1. Science 1992;255:1125-1127. 6. Volpes R, Van den Oord JJ, Desmet VJ. Immunohistochemical study of adhesion molecules in liver inflammation. HEPATOLOGY 1990;12:59-65. 7. Volpes R, Van den Oord JJ, Desmet VJ. Hepatic expression of intercellular adhesion molecule-1 (ICAM-1) in viral hepatitis B. HEPATOLOGY 1990;12:148-154. 8. Malizia G, Dino O, Pisa R, Caltagirone M, Giannuoli G, Marco VD, Aragona E, et al. Expression of leukocyte adhesion molecule in the liver of patients with chronic hepatitis B virus infection. Gastroenterology 1991;100:749-755. 9. Adams DH, Hubscher SG, Shaw J, Johnson GD, Babbs C, Rothlein R, Neuberger JM. Increased expression of intercellular adhesion molecule-1 on bile duct in primary biliary cirrhosis and primary sclerosing cholangitis. HEPATOLOGY 1991;14:426-431. 10. Volpes R, Van den Oord JJ, Desmet VJ. Vascular adhesion molecule in acute and chronic liver inflammation. HEPATOLOGY 1992;15:269-275.
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