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* Division of Trauma Surgery and
Department of Pathology, University Hospital Zurich, CH-8091 Zurich, Switzerland; and
Department of Biochemical Pharmacology, University of Konstanz, D-78457 Konstanz, Germany
1Correspondence: Division of Trauma Surgery, University Hospital Zurich, Raemistr. 100, CH-8091 Zurich, Switzerland. E-mail: guido.wanner{at}chi.usz.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: liver failure • apoptosis • hepatic microcirculation • cysteine protease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. The overall mortality of patients
suffering from acute liver failure, irrespective of the etiology, is
between 70% and 80% (4
, 5)
. Liver dysfunction/failure may be induced
by direct damage of hepatocytes through toxic agents or by
deterioration of the hepatic microcirculation in the course of
inflammatory reactions leading to local hypoxia and secondary
parenchymal cell death. Apoptosis is a major form of cell death in
hepatocytes and plays a physiological role in maintaining liver
homeostasis (6
7
8)
. However, excessive activation of the apoptotic
machinery under stressful conditions increasingly is recognized as a
pivotal mechanism in the pathophysiology of liver diseases (9
10
11
12
13
14
15
16
17
18
19)
.
CD95 (Fas/Apo1) is a member of the tumor necrosis factor/nerve growth
factor receptor superfamily and mediates apoptosis in various tissues
including liver, heart, lung, thymus, and kidney (20
, 21)
. CD95 exists
both in a soluble
(sCD95)2
and a membrane-bound form (22
, 23)
. The latter is characterized by a
transmembrane domain (`death domain`) that transduces the apoptotic
signal (24
25
26)
. CD95-induced apoptosis is mediated by an intracellular
enzyme cascade of aspartate-specific cysteine proteases, i.e.,
caspases, which is activated via the death domain (27
28
29
30
31
32)
.
The liver is highly sensitive to CD95-mediated apoptosis. In murine
experiments, systemic administration of an agonistic anti-CD95 antibody
(aCD95) (9
, 33)
caused fulminant hepatitis and acute liver failure with
death of the animals within a few hours. Histological analysis
demonstrated massive apoptotic death of hepatocytes. Full protection of
the liver was achieved by administration of the tripeptide z-VAD-fmk, a
broad caspase inhibitor with a high cell permeability, confirming the
central role of caspase activity for CD95-mediated hepatitis in
vivo (34
35
36)
. The susceptibility of the liver to aCD95-induced
acute hepatitis in mice has been attributed to a high level of
constitutive CD95 expression on the surface of hepatocytes (9
, 33
, 37
, 38)
. However, direct induction of apoptosis in individual hepatocytes
cannot fully explain the rapid destruction of the hepatic
microarchitecture observed in tissue sections of these animals (9
, 33)
.
Most recent studies demonstrate that CD95 is constitutively expressed
on sinusoidal lining cells such as endothelial cells and Kupffer cells
(KC), and is rapidly inducible under inflammatory conditions (14)
.
These findings emphasize a pivotal role of hepatic endothelial cells
and KC in the process of CD95-mediated liver failure.
Though several recent studies focused on the direct effect of CD95
activation on hepatocellular apoptosis using histological and
biochemical techniques (9
, 33
, 35)
, the implication of the hepatic
microvasculature, particularly the role of sinusoidal endothelial
cells, KC, and circulating leukocytes in vivo has not been
investigated. Little is known about the mechanisms of CD95-induced
apoptosis of nonparenchymal cells in the liver (14
, 39)
and its effect
on parenchymal cells, including the role of caspase activation.
Therefore, this study quantitatively analyzes in vivo
hepatic sinusoidal perfusion, KC activity, and leukocyte–endothelial
interaction in the course of CD95-mediated fulminant hepatitis. The
role of caspases for hepatic microcirculatory failure is assessed
in vivo using the specific inhibitor z-VAD-fmk.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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41
42
43
44
45)
.
Intravital fluorescence microscopy
Hepatic microcirculation was analyzed by epi-illumination
technique by using a modified Zeiss-Axiovert microscope with a 100-W
HBO mercury lamp (Zeiss, Jena, Germany) attached to a blue filter
system (450–490 nm/> 520 nm, excitation/emission wavelength). The
microscopic images were recorded by a CCD video camera (FK 6990, COHU,
Prospective Measurements Inc., San Diego, Calif.) and transferred to a
video system (S-VHS Panasonic AG 7350, Matsushita, Tokyo, Japan). A
final magnification of x730 was achieved on the video screen (PVM-1444
QM, Sony, Zurich, Switzerland) using a x25 objective (W 25 x/0.5;
Zeiss, Jena, Germany). Contrast enhancement was achieved by i.v.
injection of sodium fluorescein (2 µmol/kg i.v.; Merck, Darmstadt,
Germany) and allowed analysis of sinusoidal perfusion (40
41
42
, 45)
. The
use of a green filter system (530–560/> 580 nm) allowed the analysis
of leukocyte–endothelium cell interaction after staining the
leukocytes in vivo with rhodamine 6G (2 µmol/kg i.v.;
Merck) (41
, 42)
. For intravital microscopic analysis of Kupffer cell
activity, plain fluorescent latex particles (diameter 1.1 µM;
Polyscience Inc., Warrington, Pa.) were injected intra-arterially
through the femoral catheter (1.5 x 108/kg
in 0.3 ml isotonic saline) at the end of each experiment (42
43
44)
.
Quantitative video analysis
Quantitative assessment of microcirculatory parameters was
performed off-line by frame-to-frame analysis of the videotaped images.
Within 10–15 acini per animal, sinusoidal perfusion failure was
determined by counting the number of nonperfused sinusoids (given as
percentage of all sinusoids visible) (42)
. Leukocyte–endothelial cell
interaction was analyzed within 10–15 hepatic acini and 10
postsinusoidal venules per animal, including 1) the number
of stagnant leukocytes located within sinusoids (cells/lobule) and not
moving during an observation period of 20 s, as well as
2) the number of adherent leukocytes located within
postsinusoidal venules (cells/mm2 endothelial
surface, calculated from diameter and length of the vessel segment
studied, assuming cylindrical geometry) and not moving or detaching
from the endothelial lining during an observation period of 20 s (41
, 42)
.
To assess KC activity (i.e., kinetics of particle
adherence/phagocytosis), 10–15 observation fields per animal were
analyzed successively within 5 min after injection. The kinetics of KC
activity were quantified by determining the number of particles moving
in sinusoids as the percentage of all particles visible in the acini
during an observation period of 10 s (42
43
44)
. Because variations
in absolute number of particles per acinus were found in association
with alterations of sinusoidal perfusion, these data were normalized
and expressed as the percentage of particles visible in sinusoids per
microscopic field.
Experimental protocols
The animals were injected with either a hamster anti-mouse
anti-CD95 antibody (aCD95; Jo2; Pharmingen, San Diego, Calif.; 10
µg/mouse in 0.9% saline i.v.; n=24) or unspecific
immunoglobulin (IgG) (control; 10 µg/mouse in 0.9% saline i.v.;
n=24). Six animals of each group were injected i.v. with
0.25 mg z-VAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone dissolved in
DMSO; Bachem, Bubendorf, Switzerland) 5 min after application of aCD95
(aCD95/z-VAD) or IgG (control/z-VAD), respectively. Repetitive doses of
0.1 mg z-VAD-fmk were injected at 1, 2, and 3 h after aCD95 or IgG
application. Fluorescence microscopic analysis of the hepatic
microcirculation, including leukocyte–endothelial cell interaction,
sinusoidal perfusion, and KC activity, was performed 2 h and
6 h after injection of aCD95 or IgG. Separate animals were used to
study the two different time points.
Arterial blood samples were collected by cardiac puncture into heparinized syringes at the end of each experiment and centrifuged. Plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by means of standard spectrophotometric procedures.
Liver tissue specimens were obtained at the end of each experiment. Pieces of the liver were frozen at -80°C for caspase activity measurements; similar parts of each liver were obtained for light and electron microscopy and processed as described below.
Determination of caspase-3-like activity
Cytosolic extracts from liver tissue were prepared by Dounce
homogenization in hypotonic extraction buffer (25 mM HEPES, pH 7.5, 5
mM MgCl2, 1 mM EGTA, 1 mM Pefablock, and 1
µg/ml each of pepstatin, leupeptin, and aprotinin) and subsequently
centrifuged (15 min, 14,000 g, 4°C). The clear supernatant was
subsequently diluted 1:5 in extraction buffer and stored at -80°C.
Recombinant caspase-3 was diluted in glycerol buffer (50 mM HEPES, pH
7.4, 1% sucrose, 0.1% CHAPS, 20% (v/v) glycerol) and stored at
-80°C.
The fluorometric DEVD-afc cleavage assay was performed on microtiter
plates (Greiner, Nürtingen, Germany) according to the method
described by Thornberry (46)
. Cytosolic extracts (10 µl, ~1 mg/ml
protein) or recombinant caspase-3 (10 µl, 30 ng/ml protein) were
diluted 1:10 with substrate buffer (55 µM fluorogenic substrate
DEVD-afc in 50 mM HEPES, pH 7.4, 1% sucrose, 0.1% CHAPS, 10 mM DTT or
5 mM glutathione for recombinant caspase-3). Blanks contained 10 µl
extraction buffer and 90 µl substrate buffer. Generation of free afc
at 37°C was determined by fluorescence measurement at t=0/t=30 min,
using the fluorometer plate reader SLT Fluostar (SLT, Crailsheim,
Germany) set at an excitation wavelength of 385 nm and an emission
wavelength of 505 nm. Protein concentrations of the corresponding
samples were estimated with the Pierce assay (Pierce, Ill.) and the
activity was calculated using serially diluted standards (0–5 µM
afc). Control experiments confirmed that the activity was linear with
time and protein concentration under the conditions described above.
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (DEVD-afc)
and Pefablock were purchased from Biomol (Hamburg, Germany). All other
reagents not further specified were purchased from Sigma (Deisenhofen,
Germany).
Histology
For light microscopy, liver samples were fixed in 10% formalin
and embedded in paraffin. Five-micrometer sections were cut and stained
with hematoxylin and eosin. For in situ staining of
apoptotic cells, the TdT-catalyzed DNA nick end labeling (TUNEL) method
was performed using a commercially available kit (Boehringer, Mannheim,
Germany) according to the manufacturer's instructions. Sections were
counterstained with hemalaun.
For transmission electron microscopy (TEM), hepatic tissue specimens were cut into 1 mm3 cubes and fixed in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). The samples were stored in fixative for 2–3 days prior to further processing. Specimens were postfixed with osmium tetroxide, dehydrated in graded alcohol, and embedded in Epon. Ultrathin sections (60–80 nm) were cut on a Reichert ultramicrotome and contrasted with uranyl acetate and lead citrate for TEM. Stained sections were reviewed in a Philips CM 10 electron microscope operating at KV 60.
Statistical analysis
All data are expressed as means ± SE. After
disproving the assumption of normality and equal variance across
groups, differences between groups were assessed using the
Kruskal-Wallis one-way analysis of variance on ranks (overall
differences), followed by the Student-Newman-Keuls method (pairwise
multiple comparisons). Overall statistical significance was set at
P<0.05. Statistics were performed using the software
package SigmaStat (Jandel Corporation, San Rafael, Calif.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hepatic microcirculation and leukocytic response
In livers of control animals, all acini were well perfused (< 5%
nonperfused sinusoids). Treatment of animals with aCD95 resulted in a
severe (P<0.01) deterioration of sinusoidal perfusion
characterized by 36.6 ± 4.8% and 33.4 ± 6.6% nonperfused
sinusoids after 2 h and 6 h, respectively (Fig. 1
A, B). Administration of z-VAD-fmk after aCD95 application
attenuated (P<0.01) sinusoidal perfusion at 2 h
(17.3 ± 3.8% nonperfused sinusoids) (Fig. 1A
) and
almost completely inhibited aCD95-mediated perfusion failure at 6 h (8.4 ± 1.0% nonperfused sinusoids) (Fig. 1B
).
|
Analysis of zonal distribution of sinusoidal perfusion failure at
2 h demonstrated that the effect of aCD95 was most pronounced in
the midzonal and pericentral regions (38.3 ± 4.4% and 49.1% ±
4.2 of nonperfused sinusoids, Fig. 2
). Whereas z-VAD-fmk failed to inhibit perfusion failure of periportal
sinusoids, perfusion was markedly (P<0.01) improved
(19.3 ± 3.5% nonperfused sinusoids) in the midzonal region and
almost completely restored (12.7 ± 4.8% nonperfused sinusoids)
(P<0.01) in the pericentral region (P<0.01)
(Fig. 2)
. To exclude a potential influence of DMSO on hepatic
microcirculation, its effect was analyzed separately. No differences
were found in sinusoidal perfusion when DMSO was repetitively
administered in doses used to dissolve z-VAD-fmk (data not shown).
|
Analysis of leukocyte–endothelial cell interaction within the hepatic microvasculature revealed no significant differences between the study groups (data not shown). The number of stagnant leukocytes in sinusoids and adherent cells in postsinusoidal venules were comparable in the control group and after aCD95 and aCD95/z-VAD-fmk application (data not shown).
Phagocytic activity of Kupffer cells
Kinetic analysis of latex particle adherence at 2 h in
control animals revealed that 36.5 ± 11.9% of the particles
visible per screen and per 10 s of observation time were
still-moving 1 min after injection. This number decreased to 27.2 ± 1.5% and 9.4 ± 4.0% 3 and 5 min after latex particle
injection (Fig. 3
). Administration of aCD95 inhibited (P<0.05) KC activity
after 2 h as indicated by 57.5 ± 5.4% and 17.1 ±
3.1% still-moving latex particles at 1 min and 5 min after injection
(Fig. 3)
. Z-VAD-fmk restored depressed phagocytic activity of KC,
although the initial delay within the first minute after injection of
latex beads was comparable to the aCD95 group (55.6 ± 10.5%
moving particles). However, after 5 min (at the end of the observation
period), the number of moving particles was reduced
(P<0.05) to 8.3 ± 2.9%, which was similar to the
control group (Fig. 3)
. Injection of z-VAD-fmk in control animals
(control/z-VAD) did not alter any of the studied microcirculatory
parameters.
|
Liver enzymes
Plasma activities of liver enzymes were elevated
(P<0.01) 2 h after aCD95 administration and increased
(P<0.01) by 56-fold (ALT) and 57-fold (AST) after 6 h
when compared to controls. This increase of liver enzymes in plasma was
completely abolished after 2 h using z-VAD-fmk. At 6 h, only
slightly elevated plasma levels were observed (Fig. 4
).
|
Caspase-3-like activity
Anti-CD95 induced a significant (P<0.01) increase of
caspase-3-like protease activity in liver tissue, with peak levels
2 h after injection (Fig. 5
) and a subsequent decline after 6 h. Caspase activity was
completely inhibited (P<0.01) by z-VAD-fmk treatment at
both time points (Fig. 5)
.
|
Histology
Lesions of focal hepatocyte apoptosis were detected 2 h after
aCD95 injection, whereas hepatic microarchitecture was maintained
(Fig. 6
A, B). In contrast, after 6 h, extensive hepatocellular
apoptosis was observed, and the architecture of the liver parenchyma
was destroyed with hemorrhagic foci and necrosis (Fig. 6C
and D). Transmission electron micrographs demonstrated loss of
integrity of sinusoidal endothelial cells with detachment from
neighboring hepatocytes as early as 1 h after aCD95 injection
(Fig. 7
B). After 2 h, nuclear condensation in endothelial cells
and progressive denudation of sinusoids with extravasation of
erythrocytes and sinusoidal congestion were observed (Fig. 7C
). Animals treated with z-VAD-fmk revealed intact hepatic
morphology, normal-appearing hepatocytes with no evidence of apoptosis
(Fig. 6E, F
), and normal sinusoidal lining of endothelial
cells (Fig. 7A
), comparable to livers of control animals
(Fig. 7A
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent animal studies (9
, 33)
demonstrated a pivotal role of the CD95
system for fulminant hepatitis associated with high mortality within
hours. In these reports, acute liver failure was explained simply as a
direct consequence of CD95-induced apoptosis of liver parenchymal
cells. Further, these studies did not provide a pathophysiological
explanation for hemorrhagic necrosis, which was also observed. The
results from the present study indicate a more complex sequela of
synergistic pathophysiological events within the hepatic sinusoids
after application of aCD95. The injection of aCD95 antibody to mice
caused severe sinusoidal perfusion failure within 2 h, which
points to a rapid dysfunction of endothelial cells. Because sinusoidal
endothelial cells like hepatocytes constitutively express CD95 (14)
,
these cells rather than hepatocytes may be the primary target of
CD95-mediated damage due to their close proximity to circulating aCD95.
In fact, transmission electron micrographs demonstrated detachment of
sinusoidal endothelial cells as early as 1 h after injection of
aCD95 and nuclear condensation after 2 h. Sinusoidal perfusion
failure due to endothelial cell injury can explain the rapid
development of areas of focal hemorrhage and secondary necrosis of
hepatocytes as described previously by Ogasawara and co-workers (33)
.
Loss of integrity of hepatic microvessels and capillary leakage may
further facilitate direct interaction of hepatocellular membrane-bound
CD95 with the circulating aCD95 antibody, secondarily aggravating
apoptosis of hepatocytes.
Clearance capacity of KC as assessed with systemically applied latex
beads was significantly suppressed as early as 2 h after
administration of aCD95. Since local macrophages play an important role
in elimination of apoptotic cells (47
, 48)
, reduced phagocytic capacity
of KC may lead to an accumulation of apoptotic cells, as observed in
previous studies (33)
. Though a disproportion between the increased
number of apoptotic cells and the phagocytic capacity of KC was
proposed as the underlying mechanism in these reports (33)
, it is more
likely from our data that a significant inhibition of KC phagocytic
capacity represents the primary pathophysiologic event. It can be
speculated that aCD95-induced apoptosis of KC may be responsible for
reduced phagocytic capacity. This hypothesis is supported by the fact
that KC constitutively express CD95 on their surface (14)
, rendering
these cells susceptible to aCD95-induced apoptosis.
Besides macrophages, immigrating neutrophils (47
, 48)
contribute to
elimination of apoptotic cells in liver tissue. Lawson and co-workers
(18)
demonstrated that apoptosis of hepatocytes represents a strong
signal promoting transmigration of neutrophils into liver tissue when
mice are primed by an antecedent inflammatory stimulus, followed by a
septic challenge. In contrast, we did not find evidence for an active
role of leukocytes after aCD95 injection, because neither accumulation
of leukocytes in sinusoids nor leukocyte–endothelial cell interactions
in postsinusoidal venules were observed. This is in line with previous
studies that in aCD95 antibody-mediated hepatitis, little inflammatory
reaction and neutrophil immigration were detected despite massive
hepatocellular apoptosis (9
, 18
, 33)
. The contradictory results may be
due to fundamental differences of the two models. Whereas the septic
model used by Lawson et al. (18)
represents a double-hit model of
sequential stress events, the injection of aCD95 is a single-hit model.
That we did not find a leukocytic reaction in the hepatic
microcirculation despite a widespread destruction of liver tissue can
be explained by two mechanisms: 1) the aCD95 antibody
induces a primary dysfunction of circulating leukocytes, or
2) early shutdown of sinusoidal perfusion prevents invasion
of leukocytes in areas of tissue injury secondary to microcirculatory
failure.
Recently, aCD95-induced fulminant hepatitis has been associated with
activation of caspases (35
, 36)
. To evaluate a potential involvement of
caspases in the loss of sinusoidal cell functions, mice were treated
with the broad spectrum caspase inhibitor z-VAD-fmk, which blocks
caspase-1, -8, -3, -4, -11, and very weakly caspase-7 (24
, 27
, 49
50
51
52
53)
.
As demonstrated in our model, blockade of the caspase cascade led to a
protection of both endothelial cell and KC functions. Consequently,
sinusoidal perfusion and clearance capacity of KC were restored by
using z-VAD-fmk. These observations confirm that caspases are involved
in aCD95-induced damage of sinusoidal endothelial cells and KC.
However, our results also offer evidence that aCD95-mediated
microvascular damage may in part be independent of caspase activation,
because z-VAD-fmk was ineffective on sinusoidal perfusion failure in
the periportal zone and did not fully prevent hypoperfusion of midzonal
sinusoids, though no caspase activity was measured in livers of these
animals. Most recent data (51)
indicate that CD95 triggering may
activate both a caspase-dependent pathway, leading to apoptosis, and a
second pathway that involves generation of oxygen radicals resulting in
necrosis. The latter becomes particularly evident in the presence of
caspase inhibitors. Although livers of animals treated with z-VAD-fmk
did not show necrotic lesions in histological sections, generation of
oxygen radicals with subsequent endothelial cell dysfunction can
explain the temporary reduction of sinusoidal perfusion soon after CD95
triggering (2 h), which recovered at 6 h. Finally, the pivotal
role of caspases for aCD95-induced lethal liver failure was supported
by the fact that z-VAD-fmk prevented death of animals after injection
of aCD95.
In summary, this study provides in vivo evidence that aCD95
antibody-mediated liver injury is a multifaceted process involving
hepatic nonparenchymal and parenchymal cells, which finally leads to
severe microvascular perfusion failure and death of hepatocytes.
Whether or not Kupffer cells and leukocytes play an active role in
initiating or propagating this process, e.g., by the release of toxic
mediators, requires further research. Blockade of excessive or
inappropriate apoptosis by repetitive injection or continuous infusion
of caspase inhibitors (52)
may represent a new therapeutic concept to
counteract acute liver dysfunction and significantly improve the
outcome. However, although toxic side effects of these substances have
not been observed (35
, 36
, 53)
, additional studies are required to
demonstrate their safety for in vivo application in
humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 4, 1999. Accepted for publication February 15, 1999.
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