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-Induced Hepatic Parenchymal Cell Apoptosis and Neutrophil-Mediated Necrosis in a Murine Endotoxin Shock Model1
*
Department of Pharmacology, Pharmacia & Upjohn, Inc., Kalamazoo, MI 49007; and
Department of Pathology, University of Texas Health Science Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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but not IL-1ß increased caspase activity and apoptosis. Gal/ET
caused neutrophil-dependent hepatocellular necrosis at 7 h (area
of necrosis, 45 ± 3%). Delayed treatment with the caspase 3-like
protease inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD)
(10 mg/kg at 3 h) attenuated apoptosis by 81 to 88% and prevented
liver cell necrosis (
5%). Z-VAD had no effect on the initial
inflammatory response, including the sequestration of neutrophils in
sinusoids. However, Z-VAD prevented neutrophil transmigration and
necrosis. Our data indicate that activation of the caspase 3 subfamily
of cysteine proteases is critical for the development of parenchymal
cell apoptosis. In addition, excessive hepatocellular apoptosis can be
an important signal for transmigration of primed neutrophils
sequestered in sinusoids. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, are critical for the pathophysiology (2, 3, 4, 5). TNF- has a
wide variety of effects on leukocytes and liver cells that support
inflammation; e.g., TNF- is one of the mediators that induce
up-regulation of the ß2 integrin Mac-1 (CD11b/CD18) on
neutrophils during endotoxemia in vivo (6); TNF- also primes Kupffer
cells (7) and neutrophils (8) for release of cytotoxic mediators. In
addition, TNF- activates the transcription factor
NF-
B4 in endothelial
cells and hepatocytes during endotoxemia (9), which leads to the
transcriptional activation of a number of proinflammatory genes, e.g.,
chemokines (10, 11), nitric oxide synthase (12, 13), and adhesion
molecules such as ICAM-1 (14, 15), VCAM-1 (16), and selectins (9, 15).
Therefore, TNF- alone or in combination with IL-1 and complement is
responsible for neutrophil sequestration in hepatic sinusoids during
endotoxemia and sepsis (14, 17). After transmigration, neutrophils
attack parenchymal cells and cause severe liver cell necrosis (14, 16, 18). Thus, TNF- is a critical early mediator for an acute
inflammatory response in the liver during endotoxemia and sepsis.
TNF- can act through binding to two different cell surface
receptors, i.e., the 55-kDa TNF-R1 and the 75-kDa TNF-R2 (19, 20). Most
of the proinflammatory effects of TNF- are mediated through TNF-R1
(20). However, TNF- can also induce apoptosis through the death
domain of TNF-R1 (21). Recently, hepatocellular apoptosis has been
characterized in various models of endotoxemia (22, 23, 24, 25), and it was
confirmed that this effect was mediated in vivo through TNF-R1 (25, 26). In all eukaryotic cells, the intracellular signal transduction
pathway leading to apoptosis involves the activation of a cascade of
cysteine proteases (caspases/ICE proteases) (27, 28, 29). Currently, there
are 10 human caspases identified (30) with equivalent enzymes in the
mouse (31). The activation of caspase 3 (CPP32)-like proteases in liver
cells was observed during the development of apoptosis after various
insults, i.e., anti-Fas Ab in vivo (32), TGF-ß1 (33),
staurosporine (33), and hypoxia-reoxygenation (34) in vitro. Moreover,
inhibitors of caspases are highly effective in preventing apoptotic
cell death (32, 33, 34). This suggests that caspase inhibitors may allow
blockage of apoptotic cell injury without affecting the TNF--induced
proinflammatory signal transduction pathway.
Although apoptotic and necrotic hepatocytes were identified during
endotoxemia (14, 15, 18, 22, 23, 24, 25, 35), the pathophysiologic significance
of hepatocellular apoptosis for the later development of liver cell
necrosis remains unclear. Previous interventions directed against
TNF- that protected against ET-induced liver injury, e.g.,
neutralizing Abs, TNF receptor knockout mice, and inhibition of TNF-
gene transcription and synthesis (2, 14, 25, 26, 36, 37), inhibited
both the inflammatory and the apoptotic response and did not allow a
conclusion regarding the relationship between apoptotic cell death and
necrosis in the liver. The beneficial effects observed with selective
blockade of adhesion molecules on neutrophils (35) and liver cells (14, 16) suggested a critical role for neutrophils in the development of
severe necrosis. Thus, apoptosis under these conditions may be an
epiphenomenon of limited relevance or could be an important signal for
neutrophil transmigration. To address this critical question, we used
the Gal/ET shock model with its extensively described inflammatory (14, 16, 18, 35, 37) and apoptotic (22, 24, 26) response to characterize the
role of caspase activation in hepatocellular apoptosis and, by using
caspase inhibitors to selectively prevent apoptosis, to study the
relevance of apoptosis for the neutrophil-induced liver cell
necrosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male mice, strains C3Heb/FeJ (ET-sensitive) and C3H/HeJ
(ET-resistant) (20–25 g body weight), were purchased from The Jackson
Laboratory (Bar Harbor, ME). The animals had free access to food
(certified rodent diet no. 5002C, PMI Feeds, Richmond, IN) and water.
The experimental protocols followed the criteria of Pharmacia & Upjohn
(Kalamazoo, MI) and of the National Research Council for the care and
use of laboratory animals in research. Animals were treated i.p. with
700 mg/kg D-Gal (Sigma Chemical Co., St. Louis, MO)
and 100 µg/kg Salmonella abortus equi ET (Sigma Chemical)
dissolved in sterile PBS (pH 7.0). Some animals were treated with
3 x 10 mg/kg of the caspase inhibitor Z-VAD (Enzyme Systems
Products, Dublin, CA); the drug was injected 3, 4.5, and 5.5 h
after Gal/ET administration. Vehicle control animals received DMSO (1
ml/kg) at the same time. Other experiments included i.v. injection of
murine recombinant TNF- (15 µg/kg; specific activity, 4 x
108 U/mg) (Genzyme, Cambridge, MA), murine rIL-1 (13
µg/kg; specific activity, 8 x 106 U/mg) (Genzyme),
or murine rIL-1ß (23 µg/kg; specific activity, 1.5 x
106 U/mg) (Genzyme) in Gal-sensitized animals.
Experimental protocols
The animals (5–8 per treatment group) were killed by cervical
dislocation various times after administration of Gal/ET or a cytokine
(TNF-, IL-1, IL-1ß). Blood was collected from the right
ventricle into a heparinized syringe and centrifuged, and plasma was
used for determination of ALT activity with Sigma test kit DG 159-UV.
Pieces of the liver were immediately homogenized for caspase activity
measurements; other parts of each liver were frozen in liquid nitrogen
and stored at -80°C for analysis of DNA fragmentation, fixed in
phosphate-buffered formalin for histologic analysis, or embedded in OCT
embedding medium (Miles Diagnostic Division, Elkhart, IN), and
snap-frozen in methylbutane cooled in liquid nitrogen for
immunohistochemistry.
Caspase activity
Freshly excised liver was homogenized in 25 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 2 mM DTT, and 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). After centrifugation at 14,000 x g, the diluted supernatant was assayed for caspase activity using synthetic fluorogenic substrates: Ac-DEVD-MCA (Ac-Asp-Glu-Val-Asp-MCA) (Peptide Institute, Inc., Osaka, Japan) for caspase 3 (CPP32)/caspase 7 (Mch3) and Ac-YVAD-MCA (Ac-Tyr-Val-Ala-Asp-MCA) (Peptide Institute) for caspase 1 (ICE) at concentrations of 50 µM. The kinetics of the proteolytic cleavage of the substrates was monitored in a fluorescence microplate reader (Fmax; Molecular Devices, Corp., Sunnyvale, CA) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The fluorescence intensity was calibrated with standard concentrations of MCA, and the caspase activity was calculated from the slope of the recorder trace and expressed in picomols per minute per mg of protein. Protein concentrations in the supernatant were assayed using the bicinchoninic acid kit (Sigma). For the inhibitor studies in vitro, Ac-DEVD-Ald, Ac-YVAD-Ald (Peptide Institute), or CrmA (Kamiya Biomedical Co., Seattle, WA) were added to the supernatant (3.3 nM-10 µM) 15 min before adding the substrate Ac-DEVD-MCA.
Histology
Formalin-fixed portions of the liver were embedded in paraffin and 5-µm-thick sections were cut. Neutrophils were stained by the AS-D chloroacetate esterase technique as described in detail (17). Neutrophils were identified by positive staining and morphology and were counted in 50 high power fields (x400) using a Nikon Labophot microscope (Nikon, Melville, NY). Only those neutrophils present within sinusoids or extravasated into the tissue were counted; generally, there are no neutrophils present in large hepatic vessels, e.g., venules, at that time (18). The percentage of necrotic area was estimated by evaluating parallel sections stained with hematoxylin and eosin. The pathologist (A.F.) performing the histologic evaluation (polymorphonuclear leukocytes, area of necrosis) was blinded as to the treatment of animals.
Apoptosis assays
Apoptotic cells were determined using an ApopTag in situ apoptosis peroxidase detection kit (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; TUNEL assay) (Oncor, Gaithersburg, MD) and a MicroProbe staining station (Fisher Scientific, Pittsburgh, PA). Frozen liver samples were cryosectioned (6 µm; two sections/sample, 150 µm apart) using a Leitz 1720 cryostat (W. Nuhsbaum, McHenry, IL) and assayed for direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Staining was conducted according to the manufacturer’s kit protocol for fresh frozen tissue samples. DNase 1 treatment of additional sections, to nick all DNA, served as positive controls at a concentration of 1 µg/ml determined after a preliminary dilution experiment (Appligene DNase 1, Oncor). This was prepared and conducted according to manufacturer’s instructions. TUNEL-stained mouse liver tissue sections were quantitated for apoptotic cells using computer-assisted image analysis and Optimas software (Bioscan, Edmonds, WA). Visual images from TUNEL-stained slides were captured with a digital camera attached to a microscope with modifications of previously published methods (38). A color threshold was set (dark brown for diaminobenzidine) to match visual recognition of apoptotic cells. Ten random microscopic regions of interest (each covering a surface area of 0.55 mm2) were evaluated per tissue slide (two slides/sample) and for the number of apoptotic cells captured into computer memory and downloaded into Excel 4.0 (Microsoft Co, Redmond, WA) spreadsheets for statistical analysis.
For the cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN), a 20% homogenate in 50 mM sodium phosphate buffer (120 mM NaCl, 10 mM EDTA) was prepared and centrifuged at 14,000 x g. Diluted supernatant was used for the ELISA. In this test, the kinetics of product generation (Vmax) is a measure of DNA fragmentation. The Vmax values obtained for untreated controls (100%) are compared with those in treated groups. The assay allows the specific quantitation of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of cell lysates and was used extensively to demonstrate apoptosis in the liver in vivo (22, 23, 26).
Isolation of mouse liver cells
Animals (n = 4 per group) were
anesthetized with a ketamine mixture (225 mg/kg ketamine; 11.4 mg/kg
xylazine; 2.3 mg/kg acepromazine) i.m. The liver was perfused free of
blood in an open system for 5 to 10 min using an oxygenated
Ca2+-free Hanks’ buffer. A collagenase-supplemented (25
mg/100 ml buffer) Hanks’ buffer was used to digest the liver. When
good digestion was obtained (
10 min), the liver was removed, minced,
and strained through a tissue sieve. Cells were then centrifuged at
50 x g for 3 min. The supernatant (nonparenchymal
cells) was removed and saved. The pellet (parenchymal cells) was
resuspended in Hanks’ buffer and spun at 50 x g for 3
min. The supernatant was combined with the supernatant from the first
spin, and the pellet was resuspended. Cell fractions were then spun at
600 x g for 10 min. The supernatants were discarded,
and the nonparenchymal pellet was resuspended in pronase buffer (200
mg/50 ml buffer) and stirred for 10 min to remove any hepatocytes in
the suspension. This solution was then spun at 600 x g
for 10 min, and the pellet was washed once. Both cell fractions
were exposed to an ammonium chloride lysing solution for 10 min to lyse
contaminating RBC. Cells were washed again, resuspended, and counted.
Cell fractions were >98% pure and >95% viable as judged by
trypan blue exclusion. Cell concentrations were adjusted to 4 x
106 cells/ml with either caspase assay buffer or 50 mM
phosphate buffer (DNA fragmentation ELISA).
Statistics
All data are given as mean ± SE. Statistical significance between the control group and a treated group was determined with the unpaired Student’s t test, or Wilcoxon rank sum test. Comparisons between multiple groups were performed with one way ANOVA followed by the Bonferroni t test. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). In contrast, no
increased protease activity was detected with the caspase 1 substrate
Ac-YVAD-MCA. These results suggest the activation of proapoptotic
caspase 3-like proteases during endotoxemia in Gal-sensitized animals.
Parallel to the increase of caspase 3-like protease activity, there was
enhanced DNA fragmentation, indicating cells undergoing apoptosis at
the same time (Fig. 1B). Neither Gal nor ET alone caused an increase in
hepatic caspase activity or DNA fragmentation (data not shown).
Apoptosis in the liver of Gal/ET-treated animals was confirmed using
the TUNEL assay (Fig. 2). Quantitative
analysis showed few apoptotic cells in controls (about 0.2% of
hepatocytes) and no significant change up to 4 h after Gal/ET
treatment (Fig. 3). However, at 5 and
6 h, the number of apoptotic cells increased dramatically reaching
13% at 6 h (65-fold increase). These data indicate that the
increase of proapoptotic caspase activity correlated with the increase
of apoptotic cell death in this model. Because the intact tissue
did not allow us to distinguish which liver cell types had been
affected, parenchymal cells (hepatocytes) and nonparenchymal cells
(mixture of Kupffer cells and endothelial cells) were isolated 5.5
h after Gal-ET administration. Measurement of DEVD-MCA caspase activity
and DNA fragmentation showed significant increases only in hepatocytes
but not in the nonparenchymal cell fraction (Fig. 4).
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). An IC50 of 16.75 nM was
calculated from these data. In contrast, the caspase 1 inhibitor
Ac-YVAD-Ald or CrmA, an effective inhibitor of caspase 1 and the
proapoptotic caspase 8 had no or very limited effects on Gal/ET-induced
protease activity in the liver. The extrapolated IC50 for
YVAD-Ald was 19.6 µM.
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). Injection of TNF-, IL-1,
or IL-1ß in combination with Gal indicated that only Gal/TNF- was
able to induce an increase in caspase 3-like protease activity and
apoptosis in ET-resistant (Table I) and in ET-sensitive animals (data
not shown). These findings suggest that ET-induced proapoptotic caspase
activation and apoptotic cell death in hepatocytes is mediated by
TNF-.
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has been generated
and TNF--mediated inflammatory responses were initiated (NF-B
activation, transcription of adhesion molecules) (2, 6, 9, 14, 16, 18),
animals either were left untreated (disease control) or were injected
with the caspase inhibitor Z-VAD in DMSO (10 mg/kg) or with DMSO alone
(1 ml/kg). Z-VAD or vehicle treatment was repeated twice at 90-min
intervals. Gal/ET administration caused severe liver injury at 7 h
as indicated by a significant increase in plasma ALT activities and
widespread hepatocellular necrosis (45% of hepatocytes) (Fig. 6A). Z-VAD, in contrast
to the vehicle DMSO, effectively inhibited liver injury. Necrotic cell
injury was almost eliminated; i.e., only few individual hepatocytes
could be identified as necrotic. In contrast, Z-VAD did not reduce the
overall number of neutrophils in the liver (Fig. 6B).
However, virtually all neutrophils (>95%) remained localized within
sinusoids. In the vehicle group, 30 to 35% of neutrophils were
extravasated. To demonstrate the effect of Z-VAD treatment on caspase
activity and apoptosis, the experiment was repeated, and the animals
were killed at 6 h (before the onset of massive hemorrhage and
necrosis). Z-VAD completely inhibited caspase 3-like protease activity
in the liver but had no effect on the baseline caspase 1 activity (Fig. 7). Z-VAD treatment also significantly
reduced the increase in apoptotic cell death as determined by two
independent criteria, DNA fragmentation (81% inhibition) and TUNEL
assay (88% reduction of apoptotic cells) (Table II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, no increase of activity was seen with YVAD-MCA, a substrate
more specific for the caspase 1 subfamily. Furthermore, the enhanced
caspase activity could be inhibited by DEVD-Ald but not by YVAD-Ald or
CrmA. These data suggest that in the Gal/ET model in vivo, there is a
predominant activation of the caspase 3-like subfamily of proteases.
Caspase activation correlated with development of hepatocellular
apoptosis in vivo. These results are consistent with critical
involvement of caspase 3 subfamily members in apoptosis in a variety of
cell types (27, 28, 29). The effect of Gal/ET treatment on hepatic caspase
activity was not observed in ET-resistant animals that do not generate
cytokines in response to ET. Furthermore, caspase activation could be
induced with TNF- but not with IL-1 in ET-resistant and -sensitive
mice. These findings strongly indicate that Gal/ET-induced activation
of caspase 3-like proteases in vivo and hepatocellular apoptosis is
mediated by TNF-. Other stimuli that induced caspase activation in
hepatocytes, e.g., Fas Ab (32), TGF-ß1 (33), and
hypoxia-reoxygenation (34), showed increased protease activity with
DEVD-MCA and YVAD-MCA as substrates. Inhibitors of caspase 1 (YVAD-Ald,
YVAD-cmk) and caspase 3 (DEVD-Ald, Z-VAD) inhibited hepatocyte
apoptosis in vitro (34, 39) and after Fas Ab administration in vivo
(32, 39). On the basis of these data, Rouquet et al. (40) suggested
that at least two distinct pathways of Fas signaling exist in
hepatocytes. Activation of caspase 1- and caspase 3-like proteases are
required, but these pathways involve different subclasses of serine
proteases and can be selectively modulated by protein tyrosine kinase
inhibitors (40). Interestingly, actinomycin/TNF--induced apoptosis
in isolated hepatocytes and in vivo could be inhibited by YVAD-cmk
(39). In contrast, our data indicated that Gal/TNF--mediated
apoptosis correlated only with increased caspase 3-like activity and
could only be inhibited with predominantly caspase 3 inhibitors, e.g.,
DEVD-Ald. This would suggest that there might also be multiple pathways
for TNF- signaling of apoptosis.
Hepatocellular apoptosis has been described in several models of ET
shock (22, 23, 24, 25, 26); however, the importance of apoptosis for the overall
injury and organ failure in these models remained unclear. Quantitative
analysis of apoptotic and necrotic parenchymal cells showed that
13% of hepatocytes could be identified as undergoing apoptosis and
only <10% of the cells were necrotic at 6 h after Gal/ET
treatment. One h later, 38 to 45% of hepatocytes were necrotic. These
data indicate that quantitively, apoptotic cell death could not explain
the three- to fourfold higher number of necrotic cells 1 h later.
Thus, apoptosis appears to be a trigger mechanism for the more severe
attack of neutrophils. This hypothesis was investigated by the use of
caspase inhibitors, which allowed the selective blockage of apoptosis.
Administration of Z-VAD in vivo inhibited caspase 3-like activity
(DEVD-MCA) but not caspase 1 activity (YVAD-MCA) and protected
effectively against Fas-mediated apoptosis (32). Furthermore, Z-VAD
blocked apoptosis (TGF-ß1, staurosporine) but not necrosis
(staurosporine) in isolated hepatocytes (41). Kinetic analysis in
hepatocyte lysate showed that Z-VAD is substantially less potent than
DEVD-Ald in inhibiting DEVD-AFC cleavage (33). However, similar
inhibition curves indicated that the mechanism of action was the same;
i.e., both inhibitors acted as suicide substrates for caspase 3 (33).
YVAD-Ald acted as a suicide substrate for caspase 1 but was a
competitive inhibitor for caspase 3 with a very high
Ki of 12.6 µM (33). Our data agree
with these findings. Whereas DEVD-Ald was a highly potent inhibitor
(IC50 of 16.75 nM) for increased caspase activity in the
liver homogenate, YVAD-Ald inhibited this activity with an
IC50 of 19.6 µM. Because Z-VAD completely prevented
the increase in hepatic caspase activity and apoptosis, it further
supports the conclusion that caspase 3-like proteases are involved in
hepatic parenchymal cell apoptosis in Gal/ET-treated animals.
Z-VAD treatment not only prevented caspase activation and apoptosis but
also suppressed liver cell necrosis. Previous studies showed that
parenchymal cell necrosis could be attenuated by antibodies against
ß2 integrins on neutrophils (35), ICAM-1 on sinusoidal
endothelial cells and hepatocytes (14) as well as VCAM-1 on sinusoidal
lining cells (16). Neutrophil transmigration in hepatic sinusoids has
been identified as a critical step for neutrophil-mediated injury in
this model (18). Antibodies to ICAM-1 or VCAM-1 blocked neutrophil
extravasation and therefore prevented liver cell necrosis (14, 16).
These data suggest that neutrophils are essential for the development
of hepatocellular necrosis in this model. Thus, TNF- initiates two
separate responses in the liver, an apoptotic response and an
inflammatory response. Treatment with Z-VAD was started at 3 h
after Gal/ET administration. Since formation of TNF- (2, 14),
neutrophil sequestration in the hepatic vasculature (18, 35),
activation of NF-B (9, 37), mRNA formation of ICAM-1 (14, 15),
VCAM-1 (16), and selectins (15), and even in part adhesion molecule
protein synthesis (16, 42) already occurs before the 3-h time point,
Z-VAD could have not affected the proinflammatory response in the
liver. The only major events of the inflammatory response that occur
after 3 h are neutrophil transmigration and the
adherence-dependent cytotoxicity against hepatocytes. The fact that
preventing apoptosis in hepatocytes suppresses neutrophil
transmigration suggests that hepatocytes undergoing apoptosis represent
a signal for neutrophil migration and attack on parenchymal cells.
Generally, apoptosis is considered a physiologic way to remove unwanted
cells without generating an inflammatory response (43, 44). In
contrast, our data suggest that apoptosis may be able to aggravate an
inflammatory response. How can these opposite viewpoints be reconciled?
First of all, our data do not argue against the fact that single-cell
apoptosis will not trigger an inflammatory response. Our data suggest
that in the presence of activated and primed neutrophils in hepatic
sinusoids, a large number of parenchymal cells undergoing apoptosis at
the same time can represent a stimulus for these leukocytes to
transmigrate and attack. Recently (45), it was reported that infection
of hepatocytes with Listeria monocytogenes causes apoptosis
and the generation of a neutrophil chemotactic factor. However, it was
unclear whether the increased neutrophil chemotaxis was actually
dependent on apoptosis or was more related to Listeria
infection. Nevertheless, this observation is consistent with the
presence of neutrophils around infected hepatocytes in vivo (45).
Neutropenia experiments in this model indicate that a major function of
neutrophils is the removal of infected hepatocytes undergoing apoptosis
(45). In the Gal/ET model, triggering the transmigration of primed
neutrophils may have a similar purpose, and this may be a general
mechanism for removal of a large number of apoptotic cells. However, as
demonstrated after Gal/ET treatment, the further activation of
neutrophils in the liver vasculature bears the risk of additional
damage to healthy tissue. Further studies are necessary to identify the
nature of the chemotactic stimulus generated by apoptotic parenchymal
cells.
In summary, a selective activation of caspase 3-like proteases was
observed in the liver that correlated with the development of apoptosis
in parenchymal cells after Gal/ET treatment. Caspase activation and
apoptosis during endotoxemia in vivo was mediated by TNF-. Injection
of Z-VAD, an effective inhibitor of the caspase 3 subfamily, prevented
caspase activation, apoptosis, and liver cell necrosis. Z-VAD did not
affect neutrophil sequestration in sinusoids but inhibited
transmigration. These data indicate that activation of the caspase 3
subfamily is critical for the development of parenchymal cell
apoptosis. In addition, excessive hepatocellular apoptosis can be an
important signal for transmigration of primed neutrophils sequestered
in sinusoids. Thus, proapoptotic caspases may be a promising
therapeutic target for ET- and sepsis-related liver failure.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hartmut Jaeschke, Department of Pharmacology (7250-300-210), Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, MI 49007. E-mail address:
3 Current address: Huntsman Cancer Institute, Salt Lake City, UT 84112.
4 Abbreviations used in this paper: NF-B, nuclear factor B; ET, endotoxin; Gal, galactosamine; ALT, alanine aminotransferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ICE, IL-1ß-converting enzyme; Z-VAD, Z-Val-Ala-Asp-CH2F; Ac-DEVD-MCA, acetyl-Asp-Glu-Val-Asp-(4-methylcoumaryl-7-amide); Ac-YVAD-MCA, acetyl-Tyr-Val-Ala-Asp-(4-methylcoumaryl-7-amide); Ac-DEVD-Ald, acetyl-Asp-Glu-Val-Asp-CHO); Ac-YVAD-Ald, acetyl-Tyr-Val-Ala-Asp-CHO; CrmA, cowpox viral serpin cytokine response modifier A; Ac-YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethylketone.
Received for publication September 9, 1997. Accepted for publication December 1, 1997.
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 Y. Wang, R. Singh, J. H. Lefkowitch, R. M. Rigoli, and M. J. Czaja Tumor Necrosis Factor-induced Toxic Liver Injury Results from JNK2-dependent Activation of Caspase-8 and the Mitochondrial Death Pathway J. Biol. Chem., June 2, 2006; 281(22): 15258 - 15267. [Abstract] [Full Text] [PDF]
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H. Jaeschke Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1083 - G1088. [Abstract] [Full Text] [PDF]
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 M. Shea-Budgell, M. Dojka, M. Nimmo, D. Lee, and Z. Xu A BRIEF COMMUNICATION: Marginal Zinc Deficiency Increased the Susceptibility to Acute Lipopolysaccharide-Induced Liver Injury in Rats. Experimental Biology and Medicine, May 1, 2006; 231(5): 553 - 558. [Abstract] [Full Text] [PDF]
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H. Jaeschke and M. L. Bajt Intracellular Signaling Mechanisms of Acetaminophen-Induced Liver Cell Death Toxicol. Sci., January 1, 2006; 89(1): 31 - 41. [Abstract] [Full Text] [PDF]
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 C. Cover, A. Mansouri, T. R. Knight, M. L. Bajt, J. J. Lemasters, D. Pessayre, and H. Jaeschke Peroxynitrite-Induced Mitochondrial and Endonuclease-Mediated Nuclear DNA Damage in Acetaminophen Hepatotoxicity J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 879 - 887. [Abstract] [Full Text] [PDF]
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T. Hasegawa, E. Malle, A. Farhood, and H. Jaeschke Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G760 - G767. [Abstract] [Full Text] [PDF]
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R. B. Dorman, J. S. Gujral, M. L. Bajt, A. Farhood, and H. Jaeschke Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia Am J Physiol Gastrointest Liver Physiol, May 1, 2005; 288(5): G880 - G886. [Abstract] [Full Text] [PDF]
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 N. Yoshimoto, S. Togo, T. Kubota, N. Kamimukai, S. Saito, Y. Nagano, I. Endo, H. Sekido, Y. Nagashima, and H. Shimada Role of transforming growth factor-{beta}1 (TGF-{beta}1) in endotoxin-induced hepatic failure after extensive hepatectomy in rats Innate Immunity, February 1, 2005; 11(1): 33 - 39. [Abstract] [PDF]
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 H. Jaeschke and M. L. Bajt Critical role of CXC chemokines in endotoxemic liver injury in mice J. Leukoc. Biol., December 1, 2004; 76(6): 1089 - 1090. [Full Text] [PDF]
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 R. Guo, Y. Wang, A. W. Minto, R. J. Quigg, and P. N. Cunningham Acute Renal Failure in Endotoxemia is Dependent on Caspase Activation J. Am. Soc. Nephrol., December 1, 2004; 15(12): 3093 - 3102. [Abstract] [Full Text] [PDF]
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E. J. Martin and P.-G. Forkert Evidence That 1,1-Dichloroethylene Induces Apoptotic Cell Death in Murine Liver J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 33 - 42. [Abstract] [Full Text] [PDF]
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J. S. Gujral, J. A. Hinson, A. Farhood, and H. Jaeschke NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G243 - G252. [Abstract] [Full Text] [PDF]
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N. C. Hoglen, L.-S. Chen, C. D. Fisher, B. P. Hirakawa, T. Groessl, and P. C. Contreras Characterization of IDN-6556 (3-{2-(2-tert-Butyl-phenylaminooxalyl)-amino]-propionylamino}-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic Acid): a Liver-Targeted Caspase Inhibitor J. Pharmacol. Exp. Ther., May 1, 2004; 309(2): 634 - 640. [Abstract] [Full Text] [PDF]
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C. Eipel, R. Bordel, R. M. Nickels, M. D. Menger, and B. Vollmar Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G769 - G776. [Abstract] [Full Text] [PDF]
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S. Malagarie-Cazenave, B. Segui, S. Leveque, V. Garcia, S. Carpentier, M.-F. Altie, A. Brouchet, V. Gouaze, N. Andrieu-Abadie, Y. Barreira, et al. Role of FAN in Tumor Necrosis Factor-{alpha} and Lipopolysaccharide-induced Interleukin-6 Secretion and Lethality in D-Galactosamine-sensitized Mice J. Biol. Chem., April 30, 2004; 279(18): 18648 - 18655. [Abstract] [Full Text] [PDF]
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A. Canbay, A. Feldstein, E. Baskin-Bey, S. F. Bronk, and G. J. Gores The Caspase Inhibitor IDN-6556 Attenuates Hepatic Injury and Fibrosis in the Bile Duct Ligated Mouse J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 1191 - 1196. [Abstract] [Full Text] [PDF]
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 D. Klintman, X. Li, and H. Thorlacius Important Role of P-Selectin for Leukocyte Recruitment, Hepatocellular Injury, and Apoptosis in Endotoxemic Mice Clin. Vaccine Immunol., January 1, 2004; 11(1): 56 - 62. [Abstract] [Full Text] [PDF]
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B. L. Copple, C. M. Rondelli, J. F. Maddox, N. C. Hoglen, P. E. Ganey, and R. A. Roth Modes of Cell Death in Rat Liver after Monocrotaline Exposure Toxicol. Sci., January 1, 2004; 77(1): 172 - 182. [Abstract] [Full Text] [PDF]
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 R. Madhotra and I.T. Gilmore Recent developments in the treatment of alcoholic hepatitis QJM, June 1, 2003; 96(6): 391 - 400. [Abstract] [Full Text] [PDF]
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 T. SCHAFER, C. SCHEUER, K. ROEMER, M. D. MENGER, and B. VOLLMAR Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death FASEB J, April 1, 2003; 17(6): 660 - 667. [Abstract] [Full Text] [PDF]
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H. Jaeschke Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G15 - G26. [Abstract] [Full Text] [PDF]
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S. B. Yee, P. E. Ganey, and R. A. Roth The Role of Kupffer Cells and TNF-{alpha} in Monocrotaline and Bacterial Lipopolysaccharide-Induced Liver Injury Toxicol. Sci., January 1, 2003; 71(1): 124 - 132. [Abstract] [Full Text] [PDF]
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Y. Takahashi, R. W. Ganster, A. Gambotto, L. Shao, T. Kaizu, T. Wu, G. P. Yagnik, A. Nakao, G. Tsoulfas, T. Ishikawa, et al. Role of NF-kappa B on liver cold ischemia-reperfusion injury Am J Physiol Gastrointest Liver Physiol, November 1, 2002; 283(5): G1175 - G1184. [Abstract] [Full Text] [PDF]
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E. Jirillo, D. Caccavo, T. Magrone, E. Piccigallo, L. Amati, A. Lembo, C. Kalis, and M. Gumenscheimer Review: The role of the liver in the response to LPS: experimental and clinical findings Innate Immunity, October 1, 2002; 8(5): 319 - 327. [Abstract] [PDF]
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J. S. Gujral, T. R. Knight, A. Farhood, M. L. Bajt, and H. Jaeschke Mode of Cell Death after Acetaminophen Overdose in Mice: Apoptosis or Oncotic Necrosis? Toxicol. Sci., June 1, 2002; 67(2): 322 - 328. [Abstract] [Full Text] [PDF]
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A. Koteish, S. Yang, H. Lin, X. Huang, and A. M. Diehl Chronic Ethanol Exposure Potentiates Lipopolysaccharide Liver Injury Despite Inhibiting Jun N-terminal Kinase and Caspase 3 Activation J. Biol. Chem., April 5, 2002; 277(15): 13037 - 13044. [Abstract] [Full Text] [PDF]
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H. Jaeschke, G. J. Gores, A. I. Cederbaum, J. A. Hinson, D. Pessayre, and J. J. Lemasters Mechanisms of Hepatotoxicity Toxicol. Sci., February 1, 2002; 65(2): 166 - 176. [Abstract] [Full Text] [PDF]
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A. Oberholzer, C. Oberholzer, F.R. Bahjat, C. K. Edwards, and L. L. Moldawer Genetic determinants of lipopolysaccharide and D-galactosamine-mediated hepatocellular apoptosis and lethality Innate Immunity, October 1, 2001; 7(5): 375 - 380. [Abstract] [PDF]
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S. Yang, H. Lin, and A. M. Diehl Fatty liver vulnerability to endotoxin-induced damage despite NF-{kappa}B induction and inhibited caspase 3 activation Am J Physiol Gastrointest Liver Physiol, August 1, 2001; 281(2): G382 - G392. [Abstract] [Full Text] [PDF]
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K. T. Alscher, P. T. Phang, T. E. McDonald, and K. R. Walley Enteral feeding decreases gut apoptosis, permeability, and lung inflammation during murine endotoxemia Am J Physiol Gastrointest Liver Physiol, August 1, 2001; 281(2): G569 - G576. [Abstract] [Full Text] [PDF]
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 S. Yang, H. Z. Lin, J. Hwang, V. P. Chacko, and A. M. Diehl Hepatic Hyperplasia in Noncirrhotic Fatty Livers: Is Obesity-related Hepatic Steatosis a Premalignant Condition? Cancer Res., July 1, 2001; 61(13): 5016 - 5023. [Abstract] [Full Text] [PDF]
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Y. G. Weiss, L. Bellin, P. K. Kim, K. M. Andrejko, C. A. Haaxma, N. Raj, E. E. Furth, and C. S. Deutschman Compensatory hepatic regeneration after mild, but not fulminant, intraperitoneal sepsis in rats Am J Physiol Gastrointest Liver Physiol, May 1, 2001; 280(5): G968 - G973. [Abstract] [Full Text] [PDF]
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 P. He, Y. Noda, and K. Sugiyama Green Tea Suppresses Lipopolysaccharide-Induced Liver Injury in D-Galactosamine-Sensitized Rats J. Nutr., May 1, 2001; 131(5): 1560 - 1567. [Abstract] [Full Text]
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C. OBERHOLZER, A. OBERHOLZER, M. CLARE-SALZLER, and L. L. MOLDAWER Apoptosis in sepsis: a new target for therapeutic exploration FASEB J, April 1, 2001; 15(6): 879 - 892. [Abstract] [Full Text] [PDF]
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K. H. N. Hoebe, R. F. Witkamp, J. Fink-Gremmels, A. S. J. P. A. M. Van Miert, and M. Monshouwer Direct cell-to-cell contact between Kupffer cells and hepatocytes augments endotoxin-induced hepatic injury Am J Physiol Gastrointest Liver Physiol, April 1, 2001; 280(4): G720 - G728. [Abstract] [Full Text] [PDF]
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 H. Fauvel, P. Marchetti, C. Chopin, P. Formstecher, and R. Neviere Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1608 - H1614. [Abstract] [Full Text] [PDF]
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A. Boulay, R. Masson, M.-P. Chenard, M. El Fahime, L. Cassard, J.-P. Bellocq, C. Sautèes-Fridman, P. Basset, and M.-C. Rio High Cancer Cell Death in Syngeneic Tumors Developed in Host Mice Deficient for the Stromelysin-3 Matrix Metalloproteinase Cancer Res., March 1, 2001; 61(5): 2189 - 2193. [Abstract] [Full Text]
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B. YANG, T. S. JOHNSON, G. L. THOMAS, P. F. WATSON, B. WAGNER, and A. M. E. NAHAS Apoptosis and Caspase-3 in Experimental Anti-Glomerular Basement Membrane Nephritis J. Am. Soc. Nephrol., March 1, 2001; 12(3): 485 - 495. [Abstract] [Full Text]
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H. Miyoshi, C. Rust, M. E. Guicciardi, and G. J. Gores NF-{{kappa}}B Is Activated in Cholestasis and Functions to Reduce Liver Injury Am. J. Pathol., March 1, 2001; 158(3): 967 - 975. [Abstract] [Full Text] [PDF]
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 R. NEVIÈRE, H. FAUVEL, C. CHOPIN, P. FORMSTECHER, and P. MARCHETTI Caspase Inhibition Prevents Cardiac Dysfunction and Heart Apoptosis in a Rat Model of Sepsis Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 218 - 225. [Abstract] [Full Text]
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 F. R. Bahjat, V. R. Dharnidharka, K. Fukuzuka, L. Morel, J. M. Crawford, M. J. Clare-Salzler, and L. L. Moldawer Reduced Susceptibility of Nonobese Diabetic Mice to TNF-{alpha} and D-Galactosamine-Mediated Hepatocellular Apoptosis and Lethality J. Immunol., December 1, 2000; 165(11): 6559 - 6567. [Abstract] [Full Text] [PDF]
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M. L. Bajt, J. A. Lawson, S. L. Vonderfecht, J. S. Gujral, and H. Jaeschke Protection against Fas Receptor-Mediated Apoptosis in Hepatocytes and Nonparenchymal Cells by a Caspase-8 Inhibitor in Vivo: Evidence for a Postmitochondrial Processing of Caspase-8 Toxicol. Sci., November 1, 2000; 58(1): 109 - 117. [Abstract] [Full Text] [PDF]
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M. Kawasaki, K. Kuwano, N. Hagimoto, T. Matsuba, R. Kunitake, T. Tanaka, T. Maeyama, and N. Hara Protection from Lethal Apoptosis in Lipopolysaccharide-Induced Acute Lung Injury in Mice by a Caspase Inhibitor Am. J. Pathol., August 1, 2000; 157(2): 597 - 603. [Abstract] [Full Text] [PDF]
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H. Hentze, F. Gantner, S. A. Kolb, and A. Wendel Depletion of Hepatic Glutathione Prevents Death Receptor-Dependent Apoptotic and Necrotic Liver Injury in Mice Am. J. Pathol., June 1, 2000; 156(6): 2045 - 2056. [Abstract] [Full Text] [PDF]
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J. A. Lawson, A. Farhood, R. D. Hopper, M. L. Bajt, and H. Jaeschke The Hepatic Inflammatory Response after Acetaminophen Overdose: Role of Neutrophils Toxicol. Sci., April 1, 2000; 54(2): 509 - 516. [Abstract] [Full Text] [PDF]
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F. Kuhnel, L. Zender, Y. Paul, M. K. Tietze, C. Trautwein, M. Manns, and S. Kubicka NFkappa B Mediates Apoptosis through Transcriptional Activation of Fas (CD95) in Adenoviral Hepatitis J. Biol. Chem., February 25, 2000; 275(9): 6421 - 6427. [Abstract] [Full Text] [PDF]
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A. A. Pahlevan, D. J. M. Wright, C. Andrews, K. M. George, P. L. C. Small, and B. M. Foxwell The Inhibitory Action of Mycobacterium ulcerans Soluble Factor on Monocyte/T Cell Cytokine Production and NF-{kappa}B Function J. Immunol., October 1, 1999; 163(7): 3928 - 3935. [Abstract] [Full Text] [PDF]
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G. A. WANNER, L. MICA, E. WANNER-SCHMID, S. A. KOLB, H. HENTZE, O. TRENTZ, and W. ERTEL Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity FASEB J, July 1, 1999; 13(10): 1239 - 1248. [Abstract] [Full Text]
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J. Li, C. A. Bombeck, S. Yang, Y.-M. Kim, and T. R. Billiar Nitric Oxide Suppresses Apoptosis via Interrupting Caspase Activation and Mitochondrial Dysfunction in Cultured Hepatocytes J. Biol. Chem., June 11, 1999; 274(24): 17325 - 17333. [Abstract] [Full Text] [PDF]
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D. H.S. Lee, J. P. Macintyre, G. R. Taylor, E. Wang, R. K. Plante, S. S.C. Tam, B. L. Pope, and C. Y. Lau Tepoxalin Enhances the Activity of an Antioxidant, Pyrrolidine Dithiocarbamate, in Attenuating Tumor Necrosis Factor alpha -Induced Apoptosis in WEHI 164 Cells J. Pharmacol. Exp. Ther., June 1, 1999; 289(3): 1465 - 1471. [Abstract] [Full Text]
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A. MIGNON, N. ROUQUET, M. FABRE, S. MARTIN, J. C. PAGES, J. F. DHAINAUT, A. KAHN, P. BRIAND, and V. JOULIN LPS Challenge in D-galactosamine-Sensitized Mice Accounts for Caspase-dependent Fulminant Hepatitis, not for Septic Shock Am. J. Respir. Crit. Care Med., April 1, 1999; 159(4): 1308 - 1315. [Abstract] [Full Text] [PDF]
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J. J. Reiners Jr. and R. E. Clift Aryl Hydrocarbon Receptor Regulation of Ceramide-induced Apoptosis in Murine Hepatoma 1c1c7 Cells. A FUNCTION INDEPENDENT OF ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR J. Biol. Chem., January 22, 1999; 274(4): 2502 - 2510. [Abstract] [Full Text] [PDF]
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C. A. Bradham, J. Plumpe, M. P. Manns, D. A. Brenner, and C. Trautwein I. TNF-induced liver injury Am J Physiol Gastrointest Liver Physiol, September 1, 1998; 275(3): G387 - G392. [Abstract] [Full Text] [PDF]
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H. H. Nietsch, M. W. Roe, J. F. Fiekers, A. L. Moore, and S. D. Lidofsky Activation of Potassium and Chloride Channels by Tumor Necrosis Factor alpha . ROLE IN LIVER CELL DEATH J. Biol. Chem., June 30, 2000; 275(27): 20556 - 20561. [Abstract] [Full Text] [PDF]
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Y. Zhao, S. Li, E. E. Childs, D. K. Kuharsky, and X.-M. Yin Activation of Pro-death Bcl-2 Family Proteins and Mitochondria Apoptosis Pathway in Tumor Necrosis Factor-alpha -induced Liver Injury J. Biol. Chem., July 13, 2001; 276(29): 27432 - 27440. [Abstract] [Full Text] [PDF]
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