Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992), pp. 645-649
Protective Effect of 16,16-Dimethyl Prostaglandin E 2 on Isolated Rat Hepatocytes Against ComplementMediated Immune Attack YOICHI KUREBAYASHI, PhD, TAKUYA IKEDA, MSc, and YUKO HONDA, BC
16,16-Dimethyl prostaglandin E2 was examined for its ability to inhibit complementmediated in vitro hepatocytolysis by an antigen-antibody reaction. In the presence of fresh rat serum as a source of complement, 5-rain culture of isolated rat hepatocytes with a monoclonal antibody against a rat liver-specific membranous antigen resulted in a marked, significant elevation in lactate dehydrogenase leakage into the culture medium. However, with heat-inactivated rat serum, such a reaction did not occur, indicating that the hepatocytolysis induced by the antibody was attributable to the membrane damaging action of complement activated by an antigen-antibody reaction. Pretreatment of the hepatocyte with I6,16-dimethyl prostaglandin E 2 significantly suppressed the cytolytic reaction induced by the antibody in a concentration-dependent manner. These results show that 16,16-dimethyl prostaglandin E2 is capable of protecting isolated rat hepatocytes against the membrane-damaging insult of activated complement. KEY WORDS: 16,16-dimethyl prostaglandin E2; complement; antigen-antibody reaction; hepatocytolysis; cytoprotection.
The concept that prostaglandins protect the gastrointestinal mucosa against a wide variety of noxious necrotizing agents independently of their antisecretory activities has been designated cytoprotection (1, 2). Although the mechanisms of gastrointestinal cytoprotection are still far from being worked out, such a concept has recently been applied to acute experimental hepatic injuries. There is increasing evidence indicating the hepatoprotective properties of prostaglandins, including 16,16-dimethyl prosManuscript received July 5, 1991; revised manuscript received December 11, 1991; accepted December 16, 1991. From the Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai l-Chome, Edogawa-ku, Tokyo 134, Japan. Address for reprint requests: Y. Kurebayashi, Department of Allergy, Immunology Unit, 18th Floor, Guy Tower, United Medical and Dental Schools of Guy's and St. Thomas' Hospitals, St. Thomas Street, London SE1 9RT, United Kingdom.
taglandin E 2 (dmPGE2), against carbon tetrachlo-
ride-, galactosamine-, or alpha-naphthylisothiocyanate-induced toxicity in rats (3-7). Although the precise mechanism whereby prostaglandins afford such protection remains to be fully elucidated, possible stabilizing or strengthening action on the liver cell membrane has been supposed to contribute to their protective properties (3, 5). Little direct evidence, however, is available on the protective action of prostaglandins on the liver cell membrane. In the present study, we established a novel in vitro model of complement-mediated liver cell membrane injury utilizing an antigen-antibody reaction between the plasma membrane of isolated rat hepatocytes and a monoclonal antibody to a rat liver-specific membranous antigen (8, 9). Furthermore, in an attempt to confirm and expand the
Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
0163-2116/92/0500-0645506.50/09 1992PlenumPublishingCorporation
645
KUREBAYASHI ET AL
2o[
prostaglandin protection of the liver cell m e m b r a n e , d m P G E 2 was examined for its ability to inhibit the c o m p l e m e n t - m e d i a t e d hepatocytolytic reaction.
200
[
MATERIALS AND METHODS Preparation of Isolated Hepatocytes. Hepatocytes were aseptically isolated from male Sprague-Dawley rats (six weeks old; Nippon SLC, Shizuoka, Japan) with a collagenase-perfusion technique (6). The viability was over 85% in all experiments, as measured by trypan blue exclusion test. The isolated hepatocytes were cultured in 0.5 ml of Williams' E medium (WE) supplemented with 10% (v/v) fetal calf serum at 1.5 x 106 cells/well in 24-well culture plates (Primaria 3847, Nippon Becton Dickinson Co., Tokyo, Japan). After a 3-hr incubation at 37~ C in 5% CO2-95% air, the medium was replaced with 0.5 ml of WE supplemented with 20% of fresh rat serum as a source of complement, in which the hepatocytes were pretreated with varied concentrations of 16,16-dimethyl prostaglandin E2 (dmPGE2; Sigma Chemical Co., St. Louis, Missouri) at 37~ C for 5 min. In some experiments, heat-inactivated (56~ C, 30 min) rat serum was used as the supplement. Induction of Hepatocytolysis. Murine ascites containing a monoclonal antibody (MoAb) to a rat liver-specific membranous antigen was aseptically prepared as described previously (8, 9). The antibody titer of the ascites was 1:32,768 by enzyme-linked immunosorbent assay (8). The ascites was filtered through a Millipore filter (SLGVL040S, Japan Millipore, Tokyo, Japan), and 50 ~1 of the filtrate was added to the medium. The medium was collected at specified time intervals and analyzed for lactate dehydrogenase (LDH) activity as an objective biochemical index of the severity of hepatocytolytic reaction using an autoanalyzer system (Hitachi-7150, Hitachi, Tokyo, Japan). Antigen-Antibody Reaction. Rat liver cell membrane was prepared and solubilized as reported previously (9). A 96-well PVC plate (Sumitomo Bakelite Co., Tokyo, Japan) was soaked with the membrane preparation (50 I~g protein/well) for 2 hr, washed twice with phosphatebuffered saline (pH 7.4, PBS) and then blocked for 1 hr with 25% Block Ace (Yukijirushi Co., Tokyo, Japan). After addition of 100 I~1of 1600-fold diluted MoAb ascites including varied concentrations of dmPGE2, the plates were incubated for 2 hr and washed three times with PBS. Peroxidase-conjugated anti-mouse IgM (Tago, Inc., Burlingame, California) was then poured to each well, and the plate was further incubated for 2 hr and washed three times with PBS. Subsequently, 100 I~1 of peroxidase substrate ABTS (Kirkegaard and Perry LAB Inc., Annapolis, Maryland) was added to each well, and the plate was incubated for 5 min. The enzyme reaction was then measured at 405 nm with Titertec Multiskan Plate Reading Spectrophotometer (Flow Lab., McLean, Virginia). All procedures were performed at 20 ~ C. Preliminary results have validated that this experimental condition yielded saturation binding of the antibody to the membrane fraction. Statistical Analysis. The statistical analysis was carried out using the multiple-comparison test for unpaired vail-
646
"~ D
150
-ia _1 100
50 0~
MoAb( - ) I
MoAb(+)
Fresh Serum
MoAb( - )
MoAb(+)
I I__lnactivate d Serum ~
Fig 1. Hepatocytolytic reaction induced by a monoclonal antibody (MoAb) against a rat liver-specific antigen. Lactate dehydrogenase activity in the medium supernatant was used as an index of the hepatocytolysis. The isolated hepatocytes (1.5 • 106 cells) were cultured with the antibody for 5 min in the medium supplemented with 20% (v/v) fresh or heat-inactivated rat serum. Each column and vertical bar represents the mean +- sE of four experiments. **P < 0.01 vs control. ates of Dunnett (10), and P values less than 0.05 were regarded as significant. RESULTS
Hepatocyte-Damaging Property of MoAb. The damaging properties of M o A b on isolated rat hepatocytes were determined in the presence and absence of complement, and the results are illustrated in Figure 1. Addition of the M o A b ascites to the culture medium supplemented with heat-inactivated rat serum failed, at least within 5 rain, to produce a significant alteration in the m e d i u m L D H activity, which was m e a s u r e d as an objective biochemical index of the hepatocytolytic reaction. In contrast, culture of isolated h e p a t o c y t e s with M o A b in the p r e s e n c e of fresh rat serum as a source of complement led to a marked, significant increase in L D H activity in the medium supernatant. In a timecourse study with fresh rat serum, the e n z y m e activity in the culture medium sharply and markedly increased in a time-related m a n n e r for 15 min after addition of the M o A b ascites into the culture system (Figure 2). Based on these results, in the following e x p e r i m e n t , the p r o t e c t i v e effect of dmPGE2 on the isolated h e p a t o c y t e was evaluated at 5 min after initiation o f the injury. Effect of dmPGE2 on Hepatocytolysis. Figure 3 shows the protective effect of d m P G E 2 on isolated Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
dmPGE2 PROTECTION AGAINST COMPLEMENT-INDUCED LIVER CELL INJURY 600
0.4
450 D
v
3
300
I
O
0)
.J
t~
150
215
510
10'.0
15'.0
Culture Time (rain)
Fig 2. Time course of MoAb-induced hepatocytolysis induced by a monoclonal antibody against a rat liver-specific antigen in the presence of complement. Lactate dehydrogenase activity in the medium supernatant was used as an index of the hepatocytolysis. Each point and vertical bar represents the mean of three experiments.
rat hepatocytes against the injury induced by addition of the MoAb ascites into the culture system in the presence of complement. Pretreatment of isolated hepatocytes with dmPGE2 at concentrations from 10 - 6 to 10 - 4 M inhibited the complementmediated hepatocytolytic reaction in a concentration-related manner, as was evidenced by smaller increases in LDH leakage into the culture medium compared to the nontreated control value. Statistically significant inhibition of LDH leakage into the
D
0
10"~
10-5
10-4
dmPGE2 (M)
Fig 4. Effect of dmPGE2 on the antigen-antibody reaction between the liver cell membrane and a monoclonal antibody against a rat liver-specific antigen. The reaction was determined utilizing an in vitro solid-phase assay. Each column and vertical bar represents the mean -+ sE of four experiments.
medium supernatant was achieved at concentrations above 10-5 M. Effect of dmPGE 2 on Antigen-Antibody Reaction.
In an attempt to evaluate a possible interference with antigen-antibody reaction, the effect of dmPGE2 on the antigen-antibody reaction between MoAb and the liver cell membrane was determined utilizing an in vitro solid-phase assay (Figure 4). The enzyme reaction measured at 405 nm reflects the amount of the antibody bound to the liver cell membrane, dmPGE2 was without an inhibitory effect on the antigen-antibody reaction at concentrations ranging from 10-6 to 10-4 M. DISCUSSION
v
.J Q _J
0
10 -6
10 -2
10-4
dmPGIE2 (M)
Fig 3. Protective effect of 16,16-dimethyl prostaglandin E 2 on the complement-mediated hepatocytolysis induced by an antigenantibody reaction. Each column and vertical bar represents the mean -- SE of four experiments. *P < 0.05, **P < 0.01 vs control. Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
Recent studies conducted in rats have shown that dmPGE2 potently inhibited liver cell necrosis induced by carbon tetrachloride, galactosamine, and alpha-naphthylisothiocyanate (3-5). Although the detailed mechanism by which dmPGE2 exerts such hepatoprotective action has not yet been fully elucidated, several hypotheses have been proposed. Stachura and coworkers have suggested that the hepatic protection afforded by dmPGE2 against galactosamine- or carbon tetrachloride-induced injury was ascribable to possible stabilization of lysosomal and plasma membranes (3, 5). Rush et al (6) reported that dmPGEz suppressed carbon tetrachloride-induced injury by inhibiting its metabolism
647
KUREBAYASHI ET AL to reactive, toxic trichloromethyl free radicals through the cytochrome P-450-dependent oxidase enzyme system. In contrast, Raheja et al (11) found in the rat that dmPGE 2 failed to produce complete protection against hepatotoxicity by acetaminophen, a xenobiotic also metabolized by the cytochrome enzyme system. More recently, Abecassis et al (12) have reported that dmPGE 2 effectively prevents the development of fulminant hepatitis induced by murine hepatitis virus infection in mice and proposed that improved lobular microcirculation plays a role in the hepatic protection afforded by this agent in this species. It thus remains controversial whether or not dmPGE 2 can exert a truly "cyto"-protective effect on the liver parenchymal cell (13). In this study, for the purpose of testing the ability of dmPGE 2 to afford a true cytomembrane protection, we made an attempt to establish a novel in vitro model of hepatocytolysis utilizing an antigenantibody reaction between the hepatocellular plasma membrane and a monoclonal antibody to a rat liver-specific membranous antigen (8, 9). It was found that 5-min culture of isolated rat hepatocytes with MoAb in the presence of complement resulted in a marked, significant increase in the medium LDH activity that was measured as a parameter of the hepatocytolytic reaction. In contrast, such a change failed to occur with heat-inactivated serum, indicating the need for complement in the hepatocytolysis. Earlier immunohistochemical and biochemical analyses have confirmed that the monoclonal antibody employed in this study reacted only with the liver cell membrane (8) and that its target antigen is an organ- and species-specific glycoprotein that resides only in the plasma membrane of the liver cell (9). These findings, taken together with our own, may validate that complement-mediated plasma membrane rupture is an essential factor responsible for the hepatocytolytic reaction in this in vitro system. Pretreatment of isolated rat hepatocytes with dmPGE2 (10-610 -4 M) significantly suppressed the complement-mediated cellular damage in a concentration-dependent manner. This finding is essentially comparable to that by Terano et al (14), who showed a direct cytoprotective effect of dmPGE 2 (10-610 -5 M) on rat gastric epithelial cells in tissue culture, and indicates that it can exert a cytoprotective effect also on the hepatocyte independently of systemic influences. Even at the highest concentration (10 -4 M), however, dmPGE 2 failed to produce
648
a complete inhibition of the cellular damage. In this study, isolated hepatocytes were prepared by a collagenase-perfusion technique. It can be speculated that such an isolation made the cellular membrane vulnerable to the damaging insult. In a separate study, we found that an intravenous injection of MoAb to rats could provoke acute hepatic injury by a mechanism similar to that in the in vitro system used here, ie, complement-mediated immune attack (16). Further biochemical and ultrastructural studies, including evaluation of its effect on the in vivo MoAb-induced injury, are needed in order to establish an exact membrane-protecting potency of dmPGE 2. Although the precise mechanism by which dmPGE 2 protected the hepatocyte against complement-mediated immune attack remains elusive, our data show that this prostaglandin analog did not affect the reaction between MoAb and the liver cell membrane in vitro. Therefore, it is unlikely that dmPGE 2 exerted its cytoprotective effect through direct interference with the antigen-antibody reaction. It also seems implausible that inactivation of complement sequence contributes to the protection, since prostaglandins, including E2-type, were shown to lack an inhibitory effect on complement activation by the antigen-antibody reaction (16). Furthermore, the cytolytic reaction employed in this study seems totally attributable to complementmediated plasma membrane damage induced by an antigen-antibody reaction, as mentioned above. It is thus most likely that dmPGE2 protects the hepatocyte by acting on the plasma membrane to enhance the resistance against the membraneattacking insult of activated complement, possibly through stabilizing or strengthening the cellular membrane. In a preliminary experiment, we have examined the effects of several compounds, including steroids, on this in vitro cytolytic reaction, but nothing was found to be effective except dmPGE2, indicating that the protection afforded by dmPGE2 was not a nonspecific phenomenon. In summary, dmPGEz is capable of protecting isolated rat hepatocytes against complementmediated immune attack induced by an antigenantibody reaction. It is strongly suggested that such a hepatoprotective property of dmPGE2 is attributable to its direct stabilizing or strengthening action on the hepatocellular plasma membrane. The current findings support the interpretation that cytomembrane protection is an important factor in Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
dmPGE2 PROTECTION AGAINST COMPLEMENT-INDUCED LIVER CELL INJURY understanding the prostaglandin protection of the liver in various pathological conditions. REFERENCES 1. Robert A: Antisecretory, antiulcer, cytoprotective and diarrheogenic properties of prostaglandins. Adv Prostaglandin Thromboxane Res 2:507-520, 1976 2. Miller TA: Protective effects of prostaglandin against gastric mucosal damage: Current knowledge and proposed mechanism. Am J Physiol 245:G601--G623, 1983 3. Stachura J, Tarnawski A, Szczudrawa J, Bogdal J, Mach T, Klimczyk B, Kirchmayer S: Cytoprotective effect of 16,16dimethyl prostaglandin E2 and some drugs on acute galactosamine-induced liver damage in rat. Folia Histochem Cytochem 9:311-318, 1980 4. Ruwart MJ, Rush BD, Friedle NM, Piper RC, Kolaja GJ: Protective effects of 16,16-dimethyl PGE2 on the liver and kidney. Prostaglandins 21:97-102, 1981 5. Stachura J, Tarnawski A, Ivey KJ, Mach T, Bogdal J, Szczudrawa J, Klimczyk B: Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in rats. Gastroenterology 81:211-217, 1981 6. Rush B, Merritt MV, Kaluzny M, Van Schoick T, Brunden MN, Ruwart M: Studies of the mechanism of the protective action of 16,16-dimethyl PGE2 in carbon tetrachloride induced acute hepatic injury in the rat. Prostaglandins 32:439455, 1986 7. Ruwart MJ, Nichols NM, Hedeen K, Rush BD, Stachura J: 16,16-dimethyl PGE2 and fatty acids protect hepatocytes against CCl4-induced damage. In Vitro Cell Dev Biol 8:450452, 1985
Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
8. Sato K, Ikeda T, Katami K, Ogawa H: Preparation of monoclonal antibody to hepatocellular membrane and its application to induction of liver cell membrane damage. Acta Pathol Jpn 35:1375-1383, 1985 9. Sato K, Ikeda T, Kawai F, Osada Y: Isolation and purification of a rat liver-specific antigen from hepatocyte membrane. J Biochem 102:933-938, 1987 10. Dunnett CW: A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 50:1096-1121, 1955 11. Raheja KL, Linscheer WG, Cho C: Failure of exogenous prostaglandin to afford complete protection against acetaminophen-induced hepatotoxicity in the rat. J Toxicol Environ Health 15:477-484, 1985 12. Abecassis M, Falk JA, Makowka L, Dindzans VJ, Falk RE, Levy GA: 16,16-Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain 3 infection. J Clin Invest 80:881-889, 1987 13. Conn HO: Hepatic cytoprotection by prostaglandins: Theories unlimited. Hepatology 8:969-978, 1988 14. Terano A, Mach T, Stachura J, Tarnawski A, Ivey KJ: Effect of 16,16 dimethyl prostaglandin E2 on aspirin induced damage to rat gastric epithelial cells in tissue culture. Gut 25:19-25, 1984 15. Ikeda T, Kurebayashi Y: A rat model of acute liver necrosis induced by a monoclonal antibody to liver-specific antigen and complement. Hepatology 13:1152-1157, 1991 16. Kunkel SL, Thrall RS, Kunkel RG, McCormick JR, Ward PA, Zurier RB: Suppression of immune complex vasculitis in rats by prostaglandin. J Clin Invest 64:1525-1529, 1979
649
Protective Effect of 16,16-Dimethyl Prostaglandin E 2 on Isolated Rat Hepatocytes Against ComplementMediated Immune Attack YOICHI KUREBAYASHI, PhD, TAKUYA IKEDA, MSc, and YUKO HONDA, BC
16,16-Dimethyl prostaglandin E2 was examined for its ability to inhibit complementmediated in vitro hepatocytolysis by an antigen-antibody reaction. In the presence of fresh rat serum as a source of complement, 5-rain culture of isolated rat hepatocytes with a monoclonal antibody against a rat liver-specific membranous antigen resulted in a marked, significant elevation in lactate dehydrogenase leakage into the culture medium. However, with heat-inactivated rat serum, such a reaction did not occur, indicating that the hepatocytolysis induced by the antibody was attributable to the membrane damaging action of complement activated by an antigen-antibody reaction. Pretreatment of the hepatocyte with I6,16-dimethyl prostaglandin E 2 significantly suppressed the cytolytic reaction induced by the antibody in a concentration-dependent manner. These results show that 16,16-dimethyl prostaglandin E2 is capable of protecting isolated rat hepatocytes against the membrane-damaging insult of activated complement. KEY WORDS: 16,16-dimethyl prostaglandin E2; complement; antigen-antibody reaction; hepatocytolysis; cytoprotection.
The concept that prostaglandins protect the gastrointestinal mucosa against a wide variety of noxious necrotizing agents independently of their antisecretory activities has been designated cytoprotection (1, 2). Although the mechanisms of gastrointestinal cytoprotection are still far from being worked out, such a concept has recently been applied to acute experimental hepatic injuries. There is increasing evidence indicating the hepatoprotective properties of prostaglandins, including 16,16-dimethyl prosManuscript received July 5, 1991; revised manuscript received December 11, 1991; accepted December 16, 1991. From the Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai l-Chome, Edogawa-ku, Tokyo 134, Japan. Address for reprint requests: Y. Kurebayashi, Department of Allergy, Immunology Unit, 18th Floor, Guy Tower, United Medical and Dental Schools of Guy's and St. Thomas' Hospitals, St. Thomas Street, London SE1 9RT, United Kingdom.
taglandin E 2 (dmPGE2), against carbon tetrachlo-
ride-, galactosamine-, or alpha-naphthylisothiocyanate-induced toxicity in rats (3-7). Although the precise mechanism whereby prostaglandins afford such protection remains to be fully elucidated, possible stabilizing or strengthening action on the liver cell membrane has been supposed to contribute to their protective properties (3, 5). Little direct evidence, however, is available on the protective action of prostaglandins on the liver cell membrane. In the present study, we established a novel in vitro model of complement-mediated liver cell membrane injury utilizing an antigen-antibody reaction between the plasma membrane of isolated rat hepatocytes and a monoclonal antibody to a rat liver-specific membranous antigen (8, 9). Furthermore, in an attempt to confirm and expand the
Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
0163-2116/92/0500-0645506.50/09 1992PlenumPublishingCorporation
645
KUREBAYASHI ET AL
2o[
prostaglandin protection of the liver cell m e m b r a n e , d m P G E 2 was examined for its ability to inhibit the c o m p l e m e n t - m e d i a t e d hepatocytolytic reaction.
200
[
MATERIALS AND METHODS Preparation of Isolated Hepatocytes. Hepatocytes were aseptically isolated from male Sprague-Dawley rats (six weeks old; Nippon SLC, Shizuoka, Japan) with a collagenase-perfusion technique (6). The viability was over 85% in all experiments, as measured by trypan blue exclusion test. The isolated hepatocytes were cultured in 0.5 ml of Williams' E medium (WE) supplemented with 10% (v/v) fetal calf serum at 1.5 x 106 cells/well in 24-well culture plates (Primaria 3847, Nippon Becton Dickinson Co., Tokyo, Japan). After a 3-hr incubation at 37~ C in 5% CO2-95% air, the medium was replaced with 0.5 ml of WE supplemented with 20% of fresh rat serum as a source of complement, in which the hepatocytes were pretreated with varied concentrations of 16,16-dimethyl prostaglandin E2 (dmPGE2; Sigma Chemical Co., St. Louis, Missouri) at 37~ C for 5 min. In some experiments, heat-inactivated (56~ C, 30 min) rat serum was used as the supplement. Induction of Hepatocytolysis. Murine ascites containing a monoclonal antibody (MoAb) to a rat liver-specific membranous antigen was aseptically prepared as described previously (8, 9). The antibody titer of the ascites was 1:32,768 by enzyme-linked immunosorbent assay (8). The ascites was filtered through a Millipore filter (SLGVL040S, Japan Millipore, Tokyo, Japan), and 50 ~1 of the filtrate was added to the medium. The medium was collected at specified time intervals and analyzed for lactate dehydrogenase (LDH) activity as an objective biochemical index of the severity of hepatocytolytic reaction using an autoanalyzer system (Hitachi-7150, Hitachi, Tokyo, Japan). Antigen-Antibody Reaction. Rat liver cell membrane was prepared and solubilized as reported previously (9). A 96-well PVC plate (Sumitomo Bakelite Co., Tokyo, Japan) was soaked with the membrane preparation (50 I~g protein/well) for 2 hr, washed twice with phosphatebuffered saline (pH 7.4, PBS) and then blocked for 1 hr with 25% Block Ace (Yukijirushi Co., Tokyo, Japan). After addition of 100 I~1of 1600-fold diluted MoAb ascites including varied concentrations of dmPGE2, the plates were incubated for 2 hr and washed three times with PBS. Peroxidase-conjugated anti-mouse IgM (Tago, Inc., Burlingame, California) was then poured to each well, and the plate was further incubated for 2 hr and washed three times with PBS. Subsequently, 100 I~1 of peroxidase substrate ABTS (Kirkegaard and Perry LAB Inc., Annapolis, Maryland) was added to each well, and the plate was incubated for 5 min. The enzyme reaction was then measured at 405 nm with Titertec Multiskan Plate Reading Spectrophotometer (Flow Lab., McLean, Virginia). All procedures were performed at 20 ~ C. Preliminary results have validated that this experimental condition yielded saturation binding of the antibody to the membrane fraction. Statistical Analysis. The statistical analysis was carried out using the multiple-comparison test for unpaired vail-
646
"~ D
150
-ia _1 100
50 0~
MoAb( - ) I
MoAb(+)
Fresh Serum
MoAb( - )
MoAb(+)
I I__lnactivate d Serum ~
Fig 1. Hepatocytolytic reaction induced by a monoclonal antibody (MoAb) against a rat liver-specific antigen. Lactate dehydrogenase activity in the medium supernatant was used as an index of the hepatocytolysis. The isolated hepatocytes (1.5 • 106 cells) were cultured with the antibody for 5 min in the medium supplemented with 20% (v/v) fresh or heat-inactivated rat serum. Each column and vertical bar represents the mean +- sE of four experiments. **P < 0.01 vs control. ates of Dunnett (10), and P values less than 0.05 were regarded as significant. RESULTS
Hepatocyte-Damaging Property of MoAb. The damaging properties of M o A b on isolated rat hepatocytes were determined in the presence and absence of complement, and the results are illustrated in Figure 1. Addition of the M o A b ascites to the culture medium supplemented with heat-inactivated rat serum failed, at least within 5 rain, to produce a significant alteration in the m e d i u m L D H activity, which was m e a s u r e d as an objective biochemical index of the hepatocytolytic reaction. In contrast, culture of isolated h e p a t o c y t e s with M o A b in the p r e s e n c e of fresh rat serum as a source of complement led to a marked, significant increase in L D H activity in the medium supernatant. In a timecourse study with fresh rat serum, the e n z y m e activity in the culture medium sharply and markedly increased in a time-related m a n n e r for 15 min after addition of the M o A b ascites into the culture system (Figure 2). Based on these results, in the following e x p e r i m e n t , the p r o t e c t i v e effect of dmPGE2 on the isolated h e p a t o c y t e was evaluated at 5 min after initiation o f the injury. Effect of dmPGE2 on Hepatocytolysis. Figure 3 shows the protective effect of d m P G E 2 on isolated Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
dmPGE2 PROTECTION AGAINST COMPLEMENT-INDUCED LIVER CELL INJURY 600
0.4
450 D
v
3
300
I
O
0)
.J
t~
150
215
510
10'.0
15'.0
Culture Time (rain)
Fig 2. Time course of MoAb-induced hepatocytolysis induced by a monoclonal antibody against a rat liver-specific antigen in the presence of complement. Lactate dehydrogenase activity in the medium supernatant was used as an index of the hepatocytolysis. Each point and vertical bar represents the mean of three experiments.
rat hepatocytes against the injury induced by addition of the MoAb ascites into the culture system in the presence of complement. Pretreatment of isolated hepatocytes with dmPGE2 at concentrations from 10 - 6 to 10 - 4 M inhibited the complementmediated hepatocytolytic reaction in a concentration-related manner, as was evidenced by smaller increases in LDH leakage into the culture medium compared to the nontreated control value. Statistically significant inhibition of LDH leakage into the
D
0
10"~
10-5
10-4
dmPGE2 (M)
Fig 4. Effect of dmPGE2 on the antigen-antibody reaction between the liver cell membrane and a monoclonal antibody against a rat liver-specific antigen. The reaction was determined utilizing an in vitro solid-phase assay. Each column and vertical bar represents the mean -+ sE of four experiments.
medium supernatant was achieved at concentrations above 10-5 M. Effect of dmPGE 2 on Antigen-Antibody Reaction.
In an attempt to evaluate a possible interference with antigen-antibody reaction, the effect of dmPGE2 on the antigen-antibody reaction between MoAb and the liver cell membrane was determined utilizing an in vitro solid-phase assay (Figure 4). The enzyme reaction measured at 405 nm reflects the amount of the antibody bound to the liver cell membrane, dmPGE2 was without an inhibitory effect on the antigen-antibody reaction at concentrations ranging from 10-6 to 10-4 M. DISCUSSION
v
.J Q _J
0
10 -6
10 -2
10-4
dmPGIE2 (M)
Fig 3. Protective effect of 16,16-dimethyl prostaglandin E 2 on the complement-mediated hepatocytolysis induced by an antigenantibody reaction. Each column and vertical bar represents the mean -- SE of four experiments. *P < 0.05, **P < 0.01 vs control. Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
Recent studies conducted in rats have shown that dmPGE2 potently inhibited liver cell necrosis induced by carbon tetrachloride, galactosamine, and alpha-naphthylisothiocyanate (3-5). Although the detailed mechanism by which dmPGE2 exerts such hepatoprotective action has not yet been fully elucidated, several hypotheses have been proposed. Stachura and coworkers have suggested that the hepatic protection afforded by dmPGE2 against galactosamine- or carbon tetrachloride-induced injury was ascribable to possible stabilization of lysosomal and plasma membranes (3, 5). Rush et al (6) reported that dmPGEz suppressed carbon tetrachloride-induced injury by inhibiting its metabolism
647
KUREBAYASHI ET AL to reactive, toxic trichloromethyl free radicals through the cytochrome P-450-dependent oxidase enzyme system. In contrast, Raheja et al (11) found in the rat that dmPGE 2 failed to produce complete protection against hepatotoxicity by acetaminophen, a xenobiotic also metabolized by the cytochrome enzyme system. More recently, Abecassis et al (12) have reported that dmPGE 2 effectively prevents the development of fulminant hepatitis induced by murine hepatitis virus infection in mice and proposed that improved lobular microcirculation plays a role in the hepatic protection afforded by this agent in this species. It thus remains controversial whether or not dmPGE 2 can exert a truly "cyto"-protective effect on the liver parenchymal cell (13). In this study, for the purpose of testing the ability of dmPGE 2 to afford a true cytomembrane protection, we made an attempt to establish a novel in vitro model of hepatocytolysis utilizing an antigenantibody reaction between the hepatocellular plasma membrane and a monoclonal antibody to a rat liver-specific membranous antigen (8, 9). It was found that 5-min culture of isolated rat hepatocytes with MoAb in the presence of complement resulted in a marked, significant increase in the medium LDH activity that was measured as a parameter of the hepatocytolytic reaction. In contrast, such a change failed to occur with heat-inactivated serum, indicating the need for complement in the hepatocytolysis. Earlier immunohistochemical and biochemical analyses have confirmed that the monoclonal antibody employed in this study reacted only with the liver cell membrane (8) and that its target antigen is an organ- and species-specific glycoprotein that resides only in the plasma membrane of the liver cell (9). These findings, taken together with our own, may validate that complement-mediated plasma membrane rupture is an essential factor responsible for the hepatocytolytic reaction in this in vitro system. Pretreatment of isolated rat hepatocytes with dmPGE2 (10-610 -4 M) significantly suppressed the complement-mediated cellular damage in a concentration-dependent manner. This finding is essentially comparable to that by Terano et al (14), who showed a direct cytoprotective effect of dmPGE 2 (10-610 -5 M) on rat gastric epithelial cells in tissue culture, and indicates that it can exert a cytoprotective effect also on the hepatocyte independently of systemic influences. Even at the highest concentration (10 -4 M), however, dmPGE 2 failed to produce
648
a complete inhibition of the cellular damage. In this study, isolated hepatocytes were prepared by a collagenase-perfusion technique. It can be speculated that such an isolation made the cellular membrane vulnerable to the damaging insult. In a separate study, we found that an intravenous injection of MoAb to rats could provoke acute hepatic injury by a mechanism similar to that in the in vitro system used here, ie, complement-mediated immune attack (16). Further biochemical and ultrastructural studies, including evaluation of its effect on the in vivo MoAb-induced injury, are needed in order to establish an exact membrane-protecting potency of dmPGE 2. Although the precise mechanism by which dmPGE 2 protected the hepatocyte against complement-mediated immune attack remains elusive, our data show that this prostaglandin analog did not affect the reaction between MoAb and the liver cell membrane in vitro. Therefore, it is unlikely that dmPGE 2 exerted its cytoprotective effect through direct interference with the antigen-antibody reaction. It also seems implausible that inactivation of complement sequence contributes to the protection, since prostaglandins, including E2-type, were shown to lack an inhibitory effect on complement activation by the antigen-antibody reaction (16). Furthermore, the cytolytic reaction employed in this study seems totally attributable to complementmediated plasma membrane damage induced by an antigen-antibody reaction, as mentioned above. It is thus most likely that dmPGE2 protects the hepatocyte by acting on the plasma membrane to enhance the resistance against the membraneattacking insult of activated complement, possibly through stabilizing or strengthening the cellular membrane. In a preliminary experiment, we have examined the effects of several compounds, including steroids, on this in vitro cytolytic reaction, but nothing was found to be effective except dmPGE2, indicating that the protection afforded by dmPGE2 was not a nonspecific phenomenon. In summary, dmPGEz is capable of protecting isolated rat hepatocytes against complementmediated immune attack induced by an antigenantibody reaction. It is strongly suggested that such a hepatoprotective property of dmPGE2 is attributable to its direct stabilizing or strengthening action on the hepatocellular plasma membrane. The current findings support the interpretation that cytomembrane protection is an important factor in Digestive Diseases and Sciences, Vol. 37, No. 5 (May 1992)
dmPGE2 PROTECTION AGAINST COMPLEMENT-INDUCED LIVER CELL INJURY understanding the prostaglandin protection of the liver in various pathological conditions. REFERENCES 1. Robert A: Antisecretory, antiulcer, cytoprotective and diarrheogenic properties of prostaglandins. Adv Prostaglandin Thromboxane Res 2:507-520, 1976 2. Miller TA: Protective effects of prostaglandin against gastric mucosal damage: Current knowledge and proposed mechanism. Am J Physiol 245:G601--G623, 1983 3. Stachura J, Tarnawski A, Szczudrawa J, Bogdal J, Mach T, Klimczyk B, Kirchmayer S: Cytoprotective effect of 16,16dimethyl prostaglandin E2 and some drugs on acute galactosamine-induced liver damage in rat. Folia Histochem Cytochem 9:311-318, 1980 4. Ruwart MJ, Rush BD, Friedle NM, Piper RC, Kolaja GJ: Protective effects of 16,16-dimethyl PGE2 on the liver and kidney. Prostaglandins 21:97-102, 1981 5. Stachura J, Tarnawski A, Ivey KJ, Mach T, Bogdal J, Szczudrawa J, Klimczyk B: Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in rats. Gastroenterology 81:211-217, 1981 6. Rush B, Merritt MV, Kaluzny M, Van Schoick T, Brunden MN, Ruwart M: Studies of the mechanism of the protective action of 16,16-dimethyl PGE2 in carbon tetrachloride induced acute hepatic injury in the rat. Prostaglandins 32:439455, 1986 7. Ruwart MJ, Nichols NM, Hedeen K, Rush BD, Stachura J: 16,16-dimethyl PGE2 and fatty acids protect hepatocytes against CCl4-induced damage. In Vitro Cell Dev Biol 8:450452, 1985
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8. Sato K, Ikeda T, Katami K, Ogawa H: Preparation of monoclonal antibody to hepatocellular membrane and its application to induction of liver cell membrane damage. Acta Pathol Jpn 35:1375-1383, 1985 9. Sato K, Ikeda T, Kawai F, Osada Y: Isolation and purification of a rat liver-specific antigen from hepatocyte membrane. J Biochem 102:933-938, 1987 10. Dunnett CW: A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc 50:1096-1121, 1955 11. Raheja KL, Linscheer WG, Cho C: Failure of exogenous prostaglandin to afford complete protection against acetaminophen-induced hepatotoxicity in the rat. J Toxicol Environ Health 15:477-484, 1985 12. Abecassis M, Falk JA, Makowka L, Dindzans VJ, Falk RE, Levy GA: 16,16-Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain 3 infection. J Clin Invest 80:881-889, 1987 13. Conn HO: Hepatic cytoprotection by prostaglandins: Theories unlimited. Hepatology 8:969-978, 1988 14. Terano A, Mach T, Stachura J, Tarnawski A, Ivey KJ: Effect of 16,16 dimethyl prostaglandin E2 on aspirin induced damage to rat gastric epithelial cells in tissue culture. Gut 25:19-25, 1984 15. Ikeda T, Kurebayashi Y: A rat model of acute liver necrosis induced by a monoclonal antibody to liver-specific antigen and complement. Hepatology 13:1152-1157, 1991 16. Kunkel SL, Thrall RS, Kunkel RG, McCormick JR, Ward PA, Zurier RB: Suppression of immune complex vasculitis in rats by prostaglandin. J Clin Invest 64:1525-1529, 1979
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