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*
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium; and
Laboratory of Vertebrate Neurobiology, The Rockefeller University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-acid glycoprotein and
1-antitrypsin, differentially prevent lethality:
1-acid glycoprotein protects in both TNF models and not
in the anti-Fas model, while 1-antitrypsin confers
protection in the TNF/D-(+)-galactosamine model only. The
protection is inversely correlated with activation of caspase-3 and
caspase-7. The data suggest that activation of caspase-3 and -7 is
essential in the in vivo induction of apoptosis leading to lethal
hepatitis and that acute phase proteins are powerful inhibitors of
apoptosis and caspase activation. Furthermore, Bcl-2 transgenic mice,
expressing Bcl-2 specifically in hepatocytes, are protected against a
lethal challenge with anti-Fas or with
TNF/D-(+)-galactosamine, but not against TNF/actinomycin D.
The acute-phase proteins might constitute an inducible
anti-apoptotic protective system, which in pathology or disturbed
homeostasis prevents excessive apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we intended to study the activation of caspases in three
experimental models of apoptosis, viz. TNF/GalN, TNF/ActD, and
anti-Fas, all three resembling acute viral hepatitis
(15, 16, 17, 18). We and others (3, 4, 5, 6) have
demonstrated that these models cause massive apoptosis of hepatocytes
of mice in vivo, followed by the release of liver transaminases. We
could also show a differential inhibition of apoptosis by acute-phase
proteins in the three models (5). We show here that
apoptosis is an early event, associated with activation of caspase-3
and -7, and that pretreatment with the acute-phase proteins
1-acid glycoprotein
(1-AGP) and
1-antitrypsin (1-AT)
prevents caspase activation and lethality. Furthermore, transgenic
overexpression of Bcl-2 protected differentially in the three models,
which suggests either different mechanisms of induction of apoptosis or
involvement of other tissues in inducing lethality in the three
models.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were obtained from Iffa Credo (Saint
Germain-sur-l’Arbresle, France) and were used at the age of 8–10 wk.
Heterozygous Bcl-2 transgenic mice were generated as follows. A
fragment containing the coding sequence of a human Bcl-2 cDNA, flanked
by a rabbit ß-globin intron and the rabbit ß-globin polyadenylation
signal (19), was inserted downstream of the human
1-AT promoter. Transgenic mice were generated
as described previously (20) using (C57BL/6 x
DBA2)F1 eggs. The highest expressor line, as
judged by Western blotting of liver extracts, was used for experiments.
Heterozygous Bcl-2 transgenic mice were crossed with C57BL/6 animals.
Transgenic offspring were identified by PCR and crossed again with
C57BL/6 to obtain 50% transgenic and 50% wild-type mice. Male and
female transgenic and nontransgenic mice were used at the age of 7–12
wk. The animals were housed in a temperature-controlled,
air-conditioned room with 12-h light/dark cycles and received water and
food ad libitum.
Reagents
Recombinant murine TNF- (TNF) was produced in
Escherichia coli and purified to homogeneity in the first
author’s laboratory. TNF had a sp. act. of 2.6 x
108 IU/mg and an endotoxin contamination of 0.07
ng/mg of protein. Endotoxin levels were assessed by a chromogenic
Limulus amebocyte lysate assay (Coatest; Chromogenix,
Stockholm, Sweden). Bovine 1-AGP, human
1-AT, ActD, and GalN were obtained from Sigma
(St. Louis, MO). Monoclonal hamster anti-mouse Fas Ab Jo2 (IgG)
(6) was purchased from PharMingen (San Diego, CA) and had
an endotoxin level of 0.05 ng/mg protein according to the
manufacturer.
Injections and blood collections
Cytokines and chemicals were dissolved in endotoxin-free PBS before use. Intraperitoneal and i.v. injections were conducted in volumes of 0.5 and 0.2 ml, respectively. Blood was taken from the retro-orbital plexus under light ether anesthesia and was allowed to clot for 30 min at 37°C and 1 h at 4°C, followed by centrifugation at 16,000 x g. Serum was stored at -20°C.
Determination of serum alanyl aminotransferase (ALT) and body temperature measurement
The ALT content was determined using an enzymatic/colorimetric kit (Sigma). Rectal body temperatures were measured with an electronic thermometer (model 2001; Comark Electronics, Littlehampton, U.K.).
Liver homogenate preparation
Livers were cut to small pieces, washed three times with glycerol buffer (10% glycerol, 5 mM EDTA, 10 mM Tris/HCl, pH 7.4, 200 mM NaCl), and homogenized with a tissue grinder (Wheaton Scientific, Millville, NJ) in the same buffer, supplemented with 1 mM PMSF, 0.3 mM aprotinin, 1 mM leupeptin, and 1 mM oxidized glutathione. All steps were performed on ice. Samples were frozen immediately at -20°C.
Assays for apoptosis
Liver homogenates were centrifuged for 20 min at 13,000 x g, and supernatant was stored at 4°C. Apoptosis was quantified by immunochemical determination of histon-complexed DNA fragments in a microtiter plate (21). Briefly, plates were coated with an Ab directed against histon H2B. After blocking, homogenates were added and a biotinylated detection Ab specific for the nucleosome subparticle of histones H2A, H2B, and DNA (22) was administered. Detection was performed with alkaline phosphatase-conjugated streptavidin (Sanvertech, Boechout, Belgium) and substrate (Sigma). Livers from PBS-treated animals were taken at 100%.
Internucleosomal DNA cleavage was studied by gel electrophoresis. DNA was prepared from 500 µl of centrifuged liver homogenate. The proteins were removed with a phenol/chloroform/isoamyl alcohol (50/48/2) and chloroform/isoamyl alcohol (24/1) extraction. DNA was precipitated overnight with 1/10 volume 5 M ammonium acetate and 9/10 volume ethanol (96%) at -20°C. DNA was resolved in Tris/EDTA, and RNA was removed by RNase H treatment. Samples were analyzed on 1.8% agarose gel and stained with ethidium bromide.
Western blotting
For Western blotting, 200 µg total protein was loaded on a 15% SDS-polyacrylamide gel. After electrophoretic separation and blotting to a nitrocellulose membrane, the different caspase fragments were detected using polyclonal Abs raised against recombinant murine caspases (23) and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Rainham, U.K.).
Fluorogenic substrate assay for caspase activity
Caspase-like activities were determined by incubation of liver
homogenate (containing 25 µg of total protein) with 50 µM of the
fluorogenic substrate
acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin (Ac-DEVD-amc)
(Peptide Institute, Osaka, Japan) in 200 µl cell-free system buffer
containing 10 mM HEPES, pH 7.4, 220 mM mannitol, 68 mM sucrose, 2 mM
NaCl, 2.5 mM KH2PO4, 0.5 mM
EGTA, 2 mM MgCl2, 5 mM pyruvate, 0.1 mM PMSF, and
1 mM DTT. The release of fluorescent amc was measured for 1 h at
2-min intervals by fluorometry (Cytofluor; PerSeptive Biosystems,
Cambridge, MA). Data are expressed as the maximal rate of increase in
fluorescence per min (
Fmax/min).
Statistics
Significant differences in DNA fragmentation, serum ALT, and body temperature were calculated using a two-tailed t test. Significant differences in final survival time were calculated using Fisher’s exact test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported the conditions needed to induce lethal
hepatitis using TNF/GalN, TNF/ActD, or anti-Fas (2, 5); lymphotoxin, in combination with GalN, also induced lethal
hepatitis, similar to TNF/GalN (our unpublished observations). Here, we
studied the induction of apoptosis and release of liver-specific
enzymes as a function of time after the administration of a lethal dose
(LD100) of TNF/GalN, TNF/ActD, TNF, or
anti-Fas. As demonstrated in Table I
,
in all three models of lethal hepatitis, apoptosis is detected by the
presence of histon-complexed DNA fragments in the liver homogenates,
before the release of ALT. Both TNF models have comparable kinetics of
events; however, induction of apoptosis in anti-Fas-treated mice
appears much faster. In these models, apparently necrosis is a
secondary phenomenon, as ALT release follows apoptosis. TNF alone, even
in very high doses, was unable to induce apoptosis, although a clear
ALT increase was observed. Neither GalN nor ActD alone had any effect
on the parameters examined (Table I).
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To verify the induction of apoptosis during lethal hepatitis, mice
were treated with a lethal dose of TNF/GalN, TNF/ActD, or anti-Fas,
livers were collected at several time points, and the extracted DNA was
analyzed on agarose gels to visualize DNA ladders, indicative of
apoptosis. We found that DNA ladders appear rather suddenly and
coinciding with the immunochemical detection of DNA fragmentation
(Figs. 1–3
and Table I
). By Western blotting and immunodetection with specific
antisera, we found that activation of procaspase-3 as well as
procaspase-7 coincided with the appearance of DNA ladder patterns. TNF,
GalN, or ActD alone had no effect (results not shown). The samples in
which caspase-3 and caspase-7 fragments were detected cleaved
fluorogenic Ac-DEVD-amc but not
acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin, which confirms that the
detected caspase-3 and caspase-7 fragments represent active caspases
(Fig. 4).
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We described earlier that 1-AGP protects
mice against lethality induced by TNF/GalN or TNF/ActD but not against
anti-Fas and that 1-AT protects only
against TNF/GalN (5, 24). In the experiments of Fig. 4, we
studied the effect of a protective dose of these acute-phase proteins
on induction of apoptosis, release of transaminases, and caspase
activation in the three hepatotoxicity models. We found that
protection against lethality correlates with the inhibition of
apoptosis, as measured by DNA fragmentation, and secondary necrosis, as
determined by ALT release. Using Ac-DEVD-amc as a substrate for
caspase-3 and caspase-7 (25, 26), we could also
demonstrate a correlation between inhibition of apoptosis and reduction
of Ac-DEVD-amc cleavage activity in the homogenates (Fig. 4), which
indicates inhibition of caspase-3 and caspase-7 activation. In Fig. 5, we confirmed that protection conferred
by both acute-phase proteins in the TNF/GalN model is also reflected in
liver DNA ladder patterns and that the activation of procaspase-3 and
procaspase-7 is inhibited. In the TNF/ActD model, only
1-AGP can inhibit the cleavage of procaspase-3
and procaspase-7. In the anti-Fas model, none of the acute-phase
proteins influence the activation of executioner caspases (data not
shown).
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It has been described that transgenic overexpression of Bcl-2 in hepatocytes of mice prevents anti-Fas-induced apoptosis of hepatocytes (27, 28). We were interested to study the effect of Bcl-2 overexpression on induction of apoptosis and lethality by the three models. In Tables II and III, we demonstrate that Bcl-2 transgenic mice are protected very well against a lethal injection of anti-Fas: hypothermia, apoptosis, transaminase release, and lethality are completely prevented. Against TNF/GalN, transgenic Bcl-2 was also able to protect, but clearly over a less broad dose range, while no protection was observed in the TNF/ActD model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-AGP and
1-AT, are able to protect in a differential
fashion, in the sense that 1-AGP protects
against the lethality induced by TNF/GalN and TNF/ActD, while
1-AT confers protection only in the former
model (2, 5, 24). We had hoped to shed more light on the
mechanism of induction of hepatitis and on the basis of the
differential protection by 1) identifying the induction of active
caspases in the hepatitis models, 2) studying the effect of the
acute-phase reactants on the activation of caspases, and 3) looking at
the effect of overproduction of Bcl-2 on the induction of acute
hepatitis. In the three hepatitis models, we first studied the kinetics of apoptotic events by determining the appearance of fragmented DNA in an ELISA system as well as on agarose gels and found that, as compared with the release of transaminases, apoptosis is an early event, followed by necrosis (which is also macroscopically visible as the livers look entirely black). The anti-Fas-induced hepatitis develops more rapidly than the TNF-induced ones. We also found that active executioner caspases-3 and -7 appear in close time relation with the appearance of ladder patterns and fragmented DNA. Clearly the strongest signals were found after anti-Fas compared with TNF/ActD and TNF/GalN, which, together with the kinetics of events, illustrates the aggressive nature of the anti-Fas model. In this latter model, similar observations were reported recently (30). We could find no active forms of other important caspases, like caspase-1, -6, or -8, perhaps because their concentrations are below detection limits. From these studies, we conclude that no fundamental differences could be found between the three models of cytokine-induced hepatitis, as far as caspases-3 and -7 are concerned, except on the level of kinetics and quantity.
We then looked at the effects of the protective acute-phase proteins,
1-AGP and 1-AT, in
the different models. We describe here that all apoptotic events (DNA
laddering, DNA fragmentation in ELISA, AST release, and activation of
caspase-3 and -7) are prevented by the acute-phase reactants in those
cases where protection against lethality is observed. These data are
remarkable not only because they demonstrate a nice correlation between
apoptosis and subsequent lethality, but also because here we see that
acute-phase protein are able to prevent the activation of caspases by
certain apoptotic triggers. Acute-phase proteins are essential
components of the acute-phase response, a fundamental reaction during
stress, trauma, and disease (reviewed in Ref. 31). In
other words, during the acute-phase response, factors are produced that
have an inherent capacity of blocking apoptosis, a process that may be
active at certain sites of inflammation, e.g., during hepatitis,
inflammatory bowel disease, etc.
Although the protection by acute-phase proteins against apoptosis per se is very interesting, we are still left with the question why this prevention is seen only in certain models and not in others. In the anti-Fas-triggered model, for instance, we were unable to see any effect of these protective proteins. We believe that the differential effect of the acute-phase proteins reflects a different mechanism of induction of apoptosis (although the executioner last-stage caspase-3 and -7 are present in all three models). We believe that the results obtained with the Bcl-2 transgenic mice illustrate this hypothesis: we described the interesting observation that liver-specific overexpression of Bcl-2 confers complete protection against anti-Fas-induced lethality, as already seen by one of us (27) and another group (28). Others were not able to find protective effects of Bcl-2 against anti-Fas, but this was in a cell-free system (32) or in an in vitro test using T cell hybridoma cells (33). We found also partial protection against TNF/GalN and no protection at all against TNF/ActD. These data may be an in vivo situation where two or perhaps three different pathways for induction of apoptosis are followed, all culminating in active caspase-3 and -7 and in lethality, but not all inhibitable by Bcl-2. The fact that different types of induction of apoptosis exist was recently shown by Scaffidi et al. (10). In analogy with their work, we could interpret our results as follows: 1) anti-Fas induces lethal apoptosis by a type 2 apoptosis, completely inhibitable by Bcl-2; 2) TNF/ActD works by a type1 apoptosis, not at all inhibitable by Bcl-2; 3) TNF/GalN uses a third pathway, perhaps consisting of elements of the former two types. Unfortunately, no further experimental data can be collected to underbuild this hypothesis. However, one should be cautious in interpreting data on Bcl-2-mediated inhibition of anti-Fas-induced apoptosis. There are indeed reports, using Bcl-2-transfected cell lines and hepatocytes in Bcl-2-transgenic mice, that describe protection by Bcl-2 against anti-Fas-induced apoptosis (27, 34, 35, 36); in contrast, data have been published showing lack of protection by Bcl-2 against anti-Fas-induced apoptosis (33, 37, 38, 39).
Additional differences between the three models of hepatitis come from
unpublished work (C. Libert) showing that in the TNF/GalN model
inflammation is present in all 18 different tissues sampled, while in
the TNF/ActD model inflammation was observed in the lung and in the
anti-Fas model only apoptosis was observed (in the liver, but also
slightly in the lung). Because acute-phase proteins such as
1-AGP are suspected to have antiinflammatory
properties, we believe that in the TNF/GalN model apoptosis is
prevented because inflammatory molecules are inhibited, some of which
contribute to the induction of apoptosis.
The mechanisms by which 1-AGP and
1-AT prevent caspase activation are unclear.
We have reported earlier that 1-AGP and
1-AT do not block TNF-induced gene expression
in hepatocytes (5). Direct inhibition of caspases upstream
of procaspase-3 and procaspase-7 is also excluded because
1-AGP and 1-AT do not
contain any caspase-inhibitory activity on the proteolytic activity of
recombinant caspases and in TNF cytotoxicity assays (our unpublished
observations). Up-regulation of Bcl-2 or Bcl-xL
by 1-AGP and/or 1-AT,
is very unlikely, because 1-AGP nor
1-AT protect in the anti-Fas model, while
Bcl-2 does (our unpublished observations). In the TNF/GalN model, we
have demonstrated the involvement of several mediators, such as PAF and
Tx-A2 (7). The induced apoptosis of hepatocytes could be
the result of a combined action of TNF, GalN, and TNF-induced
mediators. The mechanism of protection conferred by
1-AGP and 1-AT might
be the prevention of activity or release of these mediators.
Anti-Fas-induced apoptosis results from direct binding on the receptor,
without involvement of sensitizing mediators and therefore no targets
for 1-AGP or 1-AT.
The TNF/ActD model appears to behave intermediately and to involve a
mediator that can be inhibited by 1-AGP. The
inhibition of apoptosis by 1-AGP and
1-AT appears to be an early inhibition of
apoptotic mediators, and in that respect is distinct from late
interventions, such as the inhibition of apoptotic events by Hsp70,
which occurs even in the presence of active caspase-3
(40). However, the fact that acute-phase proteins are able
to prevent apoptosis in vivo, whatever the mechanism, is interesting in
view of their potential use as therapeutic agents in disorders
involving unwanted apoptosis. The acute-phase proteins might also
constitute an inducible anti-apoptotic, protective system that in
pathology or disturbed homeostasis prevents excessive apoptosis.
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Acknowledgments |
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Footnotes |
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2 W.V.M. and G.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Claude Libert, Department of Molecular Biology, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. E-mail address:
4 Abbreviations used in this paper: GalN, galactosamine; 1-AGP, 1-acid glycoprotein; 1-AT, 1-antitrypsin; Ac-DEVD-amc, acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin; ActD, actinomycin D; ALT, alanine aminotransferase; HEK, human embryonic kidney.
Received for publication March 19, 1999. Accepted for publication August 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 94:8744.1-acid glycoprotein against tumor necrosis factor-induced lethality. J. Exp. Med. 180:1571. requires transcriptional arrest. J. Immunol. 153:1778.[Abstract]
1-Acid glycoprotein and 1-antitrypsin inhibit TNF-induced but not anti-Fas-induced apoptosis of hepatocytes in mice. J. Immunol. 159:3555.[Abstract]
1) death signal. J. Biol. Chem. 273:4345.1-Antitrypsin inhibits the lethal response to TNF in mice. J. Immunol. 157:5126.[Abstract]
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