- Email: [email protected]
Hepatic stellate cells become susceptible to fas-ligand induced apoptosis during transformation to myofibroblasts
47 GENERAL
SESSION 4
piei-
1 GS4/21
REDUCTION OF KUI’FFER CELGINDUCED OXIDANT INJURY OF THE RAT IJVER BY ATRIAL NATRIURF,TIC PEPTIDE AND CYCLO-GMP RECEPTOR PROTEINS M. Bilzer’a-> H. Jaeschke’ A. M. Vollma?, G. Panmeartner’, and A. L. Gerbes’; ~Medicine II, Kliniknm Grokadern, Ludwig-Maximilians-University of Munich, Munich, Germany, Dept. of Pharmacology, Pharmacia & Upjohn, Inc., Kalamazoo, MI. and ‘Inst. of Pharmacology, Toxicology, and Pharmacy, LndwigMaximilian+University of Munich, Munich, Germany. Background/aim: The generation of reactive oxygen species (ROS) by activated Kupffer cells (KC) contributes to liver injnry following ischemia-reperfnsion and endotoxemia. Atrial natrinretic peptide (ANP) protects the liver against ischemiareperfnsion injury, suggesting a possible modulation of KC-mediated cytotoxicity by ANP. Therefore, we investigated cytoprotection by ANP during KC activation of perfused rat livers. Methods: Livers of male Sprague-Dawley rats were perfused in a nonrecirculating fashion. KC were activated by infusion of zymosan (150 pg/ml) without or with administration of ANP (2,20,200 nM), 8-bromo cyclic guanosine monophosphate (S-Br-cGMP)(SO PM) or superoxide dismntase (SOD) (50 U/ml)/Catalase (150 U/ml). Results: Activation of KC by zymosan resulted in considerable cell damage as assessed by the sinusoidal release of lactate dehydrogenase and pmine nncleoside phosphorylase. Cell damage was nearly completely prevented by SODICatalase, indicating ROS-related liver injury. ANP reduced this injnry in a concentrationdependent fashion which seemed to be mediated by the gnanylyl cyclase-coupled A receptor (CC-A receptor) and cyclic guanosine monophosphate (cGMP): expression of the GC-A receptor was found by PCR in hepatocytes, endothelial cells and Kupffer cells, and the cGMF’analogue 8-Br-&MT’ was as potent as ANP in protecting from zymosan-induced cell damage. In order to characterize the mechanism of ANP-mediated protection, several parameters were investigated. ANP 200 nM and 8-Br-cGMP significantly attenuated the prolonged increase of hepatic vascular resistance upon KC activation. Furthermore, both compounds reduced &dative cell damage following infusion of H202 (500 PM). In contrast, superoxide anion (0,‘) formation of isolated KC was not affected by ANP. Conclusions: ANP protects the liver from KC-induced cell damage. This hormonal protection is mediated via the GC-A receptor and cGMF’suggesting that cGMP receptor proteins play a critical role in preventing cell damage following oxidant stress.
ANTITUMOR
HEPATIC STELLATE CELLS BECOME SUSCEPTIBLE TO FAS-LIGAND INDUCED APOPTOSIS DURING TRANS-
FORMATION TO MYOFIBROBLASTS. A.M. Gressner. B. Lahme. S. Roth. W.R. Gong Institute of Clinical Chemistry and Pathobiochemistry, University Hospital, Pauwelsstr., 52057 Aachen, Germany
RWTH-
The expansion of the pool of myofibroblasts (MFB) is the key pathogenetic event in fibrogenesis. MFB originate from HSC in necroinflmmnatory tissue. We hypothesize that also the elimination pathway of generated MFB might be important in the process of MFB accumulation. In a first attempt, the susceptibility of HSC/MFB to soluble Fas-ligand-induced apoptosis was studied. HSC were isolated by pronasesollagenase reperfusion. Secondary cultures of HSC were established containing fully developed MFB. HSC and MFB were exposed to sFasL to measure apoptotic reactions with TUNEL reaction, immunostaining of annexin V, ELISA quantitation of nucleosomal DNA fragments, and WST-1 test, The expression levels of pro- and contra-apoptotic protooncogenes and Fas (APO-lKD95) were measured with RNAse protection assay, RT-PCR and Western blots. MFB express Fas/APO-1 but are resistant to sFasL if protein- or RNA synthesis are not inhibited. Actinomycin D and cycloheximide primed the cells to respond sensitively to sFasL induced apoptosis whereas under similar conditions HSC remained resistant. During primary culture HSC became susceptible reaching full sensitivity in the transformed stage. Protooncogene profile of MFB is characterized by a strong increase of the bax: bcl-2 ratio. It is concluded that modulation of apoptotic susceptibility of MFB could be of great relevance for fibrogenesis. FasL-sensitive MFB develop due to a dramatically increasing ratio of bax: bcl-2.
1 EFFECT
OF FIBROBLAST
(FASL) ON HEPATOCELLULAR
ENGINEERED
CARCINOMA
J&&ozdzik.
C.Oian. JJ. Lassrtc. J. Prictp
Departnnu
of
TO EXPRESS
FAS LIGAND
(HCC).
lntemnl Medicine, Faculty of Medicine, University of Navam,
Pamplona,
Spain The FssL is P type II trsnsrnzmbrmc protein and is expressed on activated T cells snd NK cells. II induce cell apoptosis in Fss expressing cells. Presence of FasL on the be some ammo cells has ken
of
thought ss one mechanism to escape immunosurvcillancc by
deletion of tllmor specific T cells. However. recent reports indicate that high expression of FuL in tnnsplantcd cells induces g&l rejection and on the other hand it has been show that tumor cells lose their tumorigenicity when they were mginencd be related to ncruitmmt
of ncutmphils by FssL with
to express FssL. This could
activationof
their cytotoxic machinery.
lesding to local inllnmmstion and grat? or tumor rejection. In this study. we have investigated antitumor effect of tibroblasts engineered to express w:
Human
FssL cDN.4
FasL on HCC in
wss cloned into pCI-nco
sn animal model.
expression vector, snd then
trmrfectcd to tibroblnst cells PA 311. Clones with stable expression of functions1 FssL were obtsincd (PA 317IFasL). Munne HCC cell line (BNL)
was used. Tumor was induced by
injection oCBNL cells subcutanously into syngcncic BalM toxicuy between PA 317IFasL and BNL was
mouse. &&:
observed.No tumor wu
receiving tbc mixture of PA 317ffasL and BNL st the ratio l:l.
A lack of direct produced in mice
25%. 85% snd 100% of
snimsls produced tumor when the ratios were 12, I:10 and 1:lOO respectively. In contrrrt PA 317 cells trmsfected with empty plssmid (PA 317/pCl) had no effect on tumor formation in all ntios. A specific immune protective c&ct
was observed in animsls receiving the
mixture of PA317iFasL and BNL cells 14 days alter disappearance of the tumor. since no tumor was prodigal? in those animals when they were rechallmged with BNL cells alone, but twuor ws produced in those mice rechallcnged with colon clnccr cells (CT 26). Afler 100 days. 50% of animals still msnifested protection. Treatment of pre-cstablishcd tuner by subcutaneous injection
of PA 317iFssL
and BNL cell
signiticaotly inhibit tumor growth, as compwd BNL
o&ore.
conclurmr:
lntmtonw
mixtore
in distal site could
with snimsls treated with PA 317/pCI sod
injection of Pa 317/FasL
had no effect on tumor gmwh.
Local expression FasL abrogate tumorigeoicity of HCC cells. Vacchtation with
the mixtwz of tibmblast expressing FasL inducing n specific mtitumonl
andhmor cells mulled in inhibitionof tumorby
immune response. These data suggest that local expression of
FssL cm be osed as a tool for immunotherapy of
CD95 LIGAND INTRABEPATIC
INDUCED
HCC.
APOPTOSIS
AS A MECHANISM
OF
IMMUNOREGULATION
Markus Miischen. Ulrich Warskulat.and Dieter Hiiussineer Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine University Diisseldorf, Moorenstr. 5, D-40225 Diisseldorf CD95 ligation plays a major role in immunoregulation by induction of apoptosis in T-lymphocytes (TLC) [l]. We studied the expression of CD95 ligand (CD95L), trammembrane (CD95tm) and soluble isoform (CD95sol) of the CD95 receptor in primary cultures of rat liver macrophages (Kupffer cells, KC), liver parenchymal cells (PC) and TLC at the mRNA level and by means of immunocytochemistry. Interferon-gamma treatment of KC led to a IO-fold increase of CD95L mRNA levels within 6 hours, which declined thereafter. In contrast, CD95L was hardly detectable in PC. Within 24hours, the number of KC staining positive for CD95L increased. As determined by the TUNEL method, apoptosis of TLC and PC was strongly induced by addition of supernatants derived from for 24 hours interferon-gamma treated KC cultures and was specific for CD95L as assessed by inhibition of the CD95-dependent apoptotic pathway. On the other band, supematants derived from interferon-gamma treated KC cultures at 48 hours exerted a protective effect on TLC and PC committed to CD95-dependent apoptosis. Compared to CD95L expression in KC, CD95sol expression occured significantly delayed upon Interferon-gamma challenge. The soluble isofonn of the CD95 receptor generated by alternative splicing has been reported to prevent TLC from CD95-mediated apoptosis [2]. The data suggest a complex and timely coordinated interplay between KC, PC and TLC with respect to interferon-gamma induced activation of the apoptotic machinery, with potential relevance for immunoregulation. These indicate a mechanism of TLC elimination in rat liver by CD95L expression in KC which appears to be self limited by subsequent upregulation of CD95sol and thus neutralization of CD95L. [l] R. Watanabe-Fukunagaet al., Nature 356: 314-317 Cheng et al., Science 263: 1759-1762 (1994)
[2] I.
(1992)
SESSION 4
piei-
1 GS4/21
REDUCTION OF KUI’FFER CELGINDUCED OXIDANT INJURY OF THE RAT IJVER BY ATRIAL NATRIURF,TIC PEPTIDE AND CYCLO-GMP RECEPTOR PROTEINS M. Bilzer’a-> H. Jaeschke’ A. M. Vollma?, G. Panmeartner’, and A. L. Gerbes’; ~Medicine II, Kliniknm Grokadern, Ludwig-Maximilians-University of Munich, Munich, Germany, Dept. of Pharmacology, Pharmacia & Upjohn, Inc., Kalamazoo, MI. and ‘Inst. of Pharmacology, Toxicology, and Pharmacy, LndwigMaximilian+University of Munich, Munich, Germany. Background/aim: The generation of reactive oxygen species (ROS) by activated Kupffer cells (KC) contributes to liver injnry following ischemia-reperfnsion and endotoxemia. Atrial natrinretic peptide (ANP) protects the liver against ischemiareperfnsion injury, suggesting a possible modulation of KC-mediated cytotoxicity by ANP. Therefore, we investigated cytoprotection by ANP during KC activation of perfused rat livers. Methods: Livers of male Sprague-Dawley rats were perfused in a nonrecirculating fashion. KC were activated by infusion of zymosan (150 pg/ml) without or with administration of ANP (2,20,200 nM), 8-bromo cyclic guanosine monophosphate (S-Br-cGMP)(SO PM) or superoxide dismntase (SOD) (50 U/ml)/Catalase (150 U/ml). Results: Activation of KC by zymosan resulted in considerable cell damage as assessed by the sinusoidal release of lactate dehydrogenase and pmine nncleoside phosphorylase. Cell damage was nearly completely prevented by SODICatalase, indicating ROS-related liver injury. ANP reduced this injnry in a concentrationdependent fashion which seemed to be mediated by the gnanylyl cyclase-coupled A receptor (CC-A receptor) and cyclic guanosine monophosphate (cGMP): expression of the GC-A receptor was found by PCR in hepatocytes, endothelial cells and Kupffer cells, and the cGMF’analogue 8-Br-&MT’ was as potent as ANP in protecting from zymosan-induced cell damage. In order to characterize the mechanism of ANP-mediated protection, several parameters were investigated. ANP 200 nM and 8-Br-cGMP significantly attenuated the prolonged increase of hepatic vascular resistance upon KC activation. Furthermore, both compounds reduced &dative cell damage following infusion of H202 (500 PM). In contrast, superoxide anion (0,‘) formation of isolated KC was not affected by ANP. Conclusions: ANP protects the liver from KC-induced cell damage. This hormonal protection is mediated via the GC-A receptor and cGMF’suggesting that cGMP receptor proteins play a critical role in preventing cell damage following oxidant stress.
ANTITUMOR
HEPATIC STELLATE CELLS BECOME SUSCEPTIBLE TO FAS-LIGAND INDUCED APOPTOSIS DURING TRANS-
FORMATION TO MYOFIBROBLASTS. A.M. Gressner. B. Lahme. S. Roth. W.R. Gong Institute of Clinical Chemistry and Pathobiochemistry, University Hospital, Pauwelsstr., 52057 Aachen, Germany
RWTH-
The expansion of the pool of myofibroblasts (MFB) is the key pathogenetic event in fibrogenesis. MFB originate from HSC in necroinflmmnatory tissue. We hypothesize that also the elimination pathway of generated MFB might be important in the process of MFB accumulation. In a first attempt, the susceptibility of HSC/MFB to soluble Fas-ligand-induced apoptosis was studied. HSC were isolated by pronasesollagenase reperfusion. Secondary cultures of HSC were established containing fully developed MFB. HSC and MFB were exposed to sFasL to measure apoptotic reactions with TUNEL reaction, immunostaining of annexin V, ELISA quantitation of nucleosomal DNA fragments, and WST-1 test, The expression levels of pro- and contra-apoptotic protooncogenes and Fas (APO-lKD95) were measured with RNAse protection assay, RT-PCR and Western blots. MFB express Fas/APO-1 but are resistant to sFasL if protein- or RNA synthesis are not inhibited. Actinomycin D and cycloheximide primed the cells to respond sensitively to sFasL induced apoptosis whereas under similar conditions HSC remained resistant. During primary culture HSC became susceptible reaching full sensitivity in the transformed stage. Protooncogene profile of MFB is characterized by a strong increase of the bax: bcl-2 ratio. It is concluded that modulation of apoptotic susceptibility of MFB could be of great relevance for fibrogenesis. FasL-sensitive MFB develop due to a dramatically increasing ratio of bax: bcl-2.
1 EFFECT
OF FIBROBLAST
(FASL) ON HEPATOCELLULAR
ENGINEERED
CARCINOMA
J&&ozdzik.
C.Oian. JJ. Lassrtc. J. Prictp
Departnnu
of
TO EXPRESS
FAS LIGAND
(HCC).
lntemnl Medicine, Faculty of Medicine, University of Navam,
Pamplona,
Spain The FssL is P type II trsnsrnzmbrmc protein and is expressed on activated T cells snd NK cells. II induce cell apoptosis in Fss expressing cells. Presence of FasL on the be some ammo cells has ken
of
thought ss one mechanism to escape immunosurvcillancc by
deletion of tllmor specific T cells. However. recent reports indicate that high expression of FuL in tnnsplantcd cells induces g&l rejection and on the other hand it has been show that tumor cells lose their tumorigenicity when they were mginencd be related to ncruitmmt
of ncutmphils by FssL with
to express FssL. This could
activationof
their cytotoxic machinery.
lesding to local inllnmmstion and grat? or tumor rejection. In this study. we have investigated antitumor effect of tibroblasts engineered to express w:
Human
FssL cDN.4
FasL on HCC in
wss cloned into pCI-nco
sn animal model.
expression vector, snd then
trmrfectcd to tibroblnst cells PA 311. Clones with stable expression of functions1 FssL were obtsincd (PA 317IFasL). Munne HCC cell line (BNL)
was used. Tumor was induced by
injection oCBNL cells subcutanously into syngcncic BalM toxicuy between PA 317IFasL and BNL was
mouse. &&:
observed.No tumor wu
receiving tbc mixture of PA 317ffasL and BNL st the ratio l:l.
A lack of direct produced in mice
25%. 85% snd 100% of
snimsls produced tumor when the ratios were 12, I:10 and 1:lOO respectively. In contrrrt PA 317 cells trmsfected with empty plssmid (PA 317/pCl) had no effect on tumor formation in all ntios. A specific immune protective c&ct
was observed in animsls receiving the
mixture of PA317iFasL and BNL cells 14 days alter disappearance of the tumor. since no tumor was prodigal? in those animals when they were rechallmged with BNL cells alone, but twuor ws produced in those mice rechallcnged with colon clnccr cells (CT 26). Afler 100 days. 50% of animals still msnifested protection. Treatment of pre-cstablishcd tuner by subcutaneous injection
of PA 317iFssL
and BNL cell
signiticaotly inhibit tumor growth, as compwd BNL
o&ore.
conclurmr:
lntmtonw
mixtore
in distal site could
with snimsls treated with PA 317/pCI sod
injection of Pa 317/FasL
had no effect on tumor gmwh.
Local expression FasL abrogate tumorigeoicity of HCC cells. Vacchtation with
the mixtwz of tibmblast expressing FasL inducing n specific mtitumonl
andhmor cells mulled in inhibitionof tumorby
immune response. These data suggest that local expression of
FssL cm be osed as a tool for immunotherapy of
CD95 LIGAND INTRABEPATIC
INDUCED
HCC.
APOPTOSIS
AS A MECHANISM
OF
IMMUNOREGULATION
Markus Miischen. Ulrich Warskulat.and Dieter Hiiussineer Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine University Diisseldorf, Moorenstr. 5, D-40225 Diisseldorf CD95 ligation plays a major role in immunoregulation by induction of apoptosis in T-lymphocytes (TLC) [l]. We studied the expression of CD95 ligand (CD95L), trammembrane (CD95tm) and soluble isoform (CD95sol) of the CD95 receptor in primary cultures of rat liver macrophages (Kupffer cells, KC), liver parenchymal cells (PC) and TLC at the mRNA level and by means of immunocytochemistry. Interferon-gamma treatment of KC led to a IO-fold increase of CD95L mRNA levels within 6 hours, which declined thereafter. In contrast, CD95L was hardly detectable in PC. Within 24hours, the number of KC staining positive for CD95L increased. As determined by the TUNEL method, apoptosis of TLC and PC was strongly induced by addition of supernatants derived from for 24 hours interferon-gamma treated KC cultures and was specific for CD95L as assessed by inhibition of the CD95-dependent apoptotic pathway. On the other band, supematants derived from interferon-gamma treated KC cultures at 48 hours exerted a protective effect on TLC and PC committed to CD95-dependent apoptosis. Compared to CD95L expression in KC, CD95sol expression occured significantly delayed upon Interferon-gamma challenge. The soluble isofonn of the CD95 receptor generated by alternative splicing has been reported to prevent TLC from CD95-mediated apoptosis [2]. The data suggest a complex and timely coordinated interplay between KC, PC and TLC with respect to interferon-gamma induced activation of the apoptotic machinery, with potential relevance for immunoregulation. These indicate a mechanism of TLC elimination in rat liver by CD95L expression in KC which appears to be self limited by subsequent upregulation of CD95sol and thus neutralization of CD95L. [l] R. Watanabe-Fukunagaet al., Nature 356: 314-317 Cheng et al., Science 263: 1759-1762 (1994)
[2] I.
(1992)