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Regular Articles |
From the Department of Anatomy,*
School of Medicine, and
Department of Retroviral Regulation,
Medical
Research Division, Tokyo Medical and Dental University, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ballooning of hepatocytes is one of the earliest, most frequent, and most conspicuous changes seen in liver injured by CCl4 administration. However, it is also observed in such conditions as viral infection, alcoholic hepatitis, biliary obstruction, starvation, choline deficiency, hypoxia, scurvy, yellow fever, and radiation injury.16 Although the relationship between the ballooning changes and necrosis and the fate of the ballooned hepatocytes have long been debated, the conventional explanation of cell ballooning is that it is a forerunner of necrosis.17,18 Thus, studies to determine the fate of the ballooned cell are viewed as important for understanding the underlying mechanisms of the aforementioned diseases.
Our objective was to re-examine the liver injury produced by the injection of CCl4 in rats and to determine whether apoptosis may coexist with necrosis as an additional underlying mechanism for toxicity. We used the analysis of DNA fragmentation by gel electrophoresis and flow cytometry to qualify and quantify apoptosis in the liver, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay to identify apoptotic cells among injured hepatocytes at the level of the single cell in situ.19,20 We further characterized the appearance time of the ballooned hepatocytes and proposed a novel mechanism in which the ballooned cells found their way by apoptosis in this model. Our evidence for the existence of apoptosis in this model actually challenges the conventional view of necrosis induced by CCl4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Wistar rats 7 to 9 weeks old were obtained from Nippon Biological Supplier (Tokyo, Japan) and were bred in the animal facilities at Tokyo Medical and Dental University. Each experimental group was composed of five rats. Animals were sacrificed by anesthesia with ether. The liver of each animal was examined at 3, 6, 12, 24, 48, 72, and 96 hours after a single intraperitoneal injection of 0.3 ml/kg of CCl4 (50% vol/vol in liquid paraffin). The control groups consisted of either untreated animals (0 hours) or those that received an injection of liquid paraffin.
Light and Electron Microscopy
The liver of each control and treated animal was perfused through the portal vein with 1.5% glutaraldehyde in 0.062 mol/L cacodylate buffer (pH 7.4) containing 1% sucrose. Blocks from the fixed tissue were immersed in 0.1 mol/L phosphate-buffered (pH 7.4) 1% osmium tetroxide for 2 hours and then dehydrated and embedded in Poly/Bed 812 (Polysciences Inc., Warrington, PA). Semithin sections (0.5 µm) were stained with toluidine blue for evaluation by light microscopy. Thin sections were doubly stained with uranyl acetate and lead citrate for examination by an electron microscope (JEOL-100 CX) operated at 100 kV.
Preparation of Samples for Flow Cytometry
Hepatocyte suspensions were prepared using the method described by Dolbeare et al21 with slight modification. Briefly, the liver was removed without perfusion. The tissue blocks were dispersed through stainless mesh in RPMI 1640 culture medium (Life Technologies, Inc., Grand Island, NY). The cell suspensions were pelleted at 500 x g for 5 minutes, fixed in 70% ethanol for 2 hours, digested in 0.05% collagenase II (Life Technologies, Inc.) at room temperature for 10 minutes, processed with 0.5 mg/ml RNase at 37°C for 20 minutes, resuspended in 50 ng/ml propidium iodide solution, and incubated for 2 hours. The stained cells were analyzed by flow cytometry using FACS Calibur (Becton Dickinson, Mountain View, CA). Data are expressed as percentage of apoptotic cells. To prevent digestion of apoptotic cells by Kupffer cells, all steps were performed at 4°C unless otherwise indicated.
DNA Gel Electrophoresis
Isolated cells were counted, pelleted, resuspended in 10 mmol/L ethylenediaminetetraacetic acid and 50 mmol/L Tris-HCl (pH 8.0) containing 0.5% sodium lauryl sarkosinate and 0.5 mg/ml proteinase K, and incubated for 60 minutes at 50°C. Then, 10 mmol/L ethylenediaminetetraacetic acid containing 0.25% bromphenol blue and 40% sucrose was mixed with each DNA extract. The individual extracts were loaded into the wells of a 2% agarose gel containing 3 µg/ml of ethidium bromide. Electrophoresis was performed in 40 mmol/L Tris-HCl containing 40 mmol/L acetic acid and 1 mmol/L ethylenediaminetetraacetic acid.
DNA TUNEL
The livers were perfused through their portal veins with saline for 20 seconds and phosphate-buffered 4% paraformaldehyde for 2 minutes. Slices from the livers were immersed in the same fixative for 6 hours at 4°C and then embedded in paraffin. Sections 4 µm thick were collected on glass slides coated with poly-L-lysine. The nuclear DNA fragmentation of apoptotic cells was labeled in situ using the TUNEL method19,20 as follows. The sections were first deparaffinized and treated for 15 minutes with 20 µg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) in 0.1 mol/L Tris-HCl buffer (pH 7.4). They were then treated with 2% H2O2 for 5 minutes and then incubated with 0.3 U/µl terminal deoxynucleotidyl transferase buffer (Life Technologies, Inc.) and 0.04 nmol/µl biotinylated dUTP (Boehringer Mannheim) in terminal deoxynucleotidyl transferase buffer (Life Technologies, Inc.) at 37°C for 60 minutes. The sections were incubated for 10 minutes with 2% bovine serum albumin followed by 30 minutes in peroxidase-conjugated streptavidin (DAKO, Carpinteria, CA) diluted 1:300 with phosphate-buffered saline. Peroxidase activity in the sections was visualized by adding 0.025% 3,3-diaminobenzidine tetrahydrochloride in 0.05 mol/L Tris-HCl buffer (pH 7.4) solution containing 0.01% H2O2 for 5 minutes. To examine the relationship between apoptotic hepatocytes and macrophages, some sections already stained by the TUNEL method were further processed for double staining (TUNEL and ED1 or ED2 or OX-6 monoclonal antibody staining) by the peroxidase anti-peroxidase complex method in a manner similar to that described above. In brief, those sections treated with the primary antibody (Ab) were successively incubated with goat immunoglobulin to mouse immunoglobulin G (DAKO) (diluted 1:50) and with mouse peroxidase anti-peroxidase complex (1:100 dilution) and then exposed to the 3,3-diaminobenzidine tetrahydrochloride solution for visualization.
Immunohistochemistry
Selected sections were immunostained using the streptavidin-biotin-peroxidase complex method with ED1, ED2 (Serotec, Kidlington, Oxford, UK), and OX-6 (Cedarlane, Hornby, Ontario, Canada) monoclonal Abs to identify Kupffer cells/macrophages and major histocompatibility complex (MHC) class II antigen (Ag)+ cells. These sections were first pretreated with 0.3% H2O2 in methanol and then incubated with normal goat serum (1:5 dilution). They were incubated overnight with ED1, ED2, or OX-6 Abs (diluted 1:1000), rinsed in phosphate-buffered saline, incubated for 30 minutes with biotinylated goat anti-mouse immunoglobulin (DAKO) (1:600 dilution), then rinsed again in phosphate-buffered saline, and finally incubated for 30 minutes with peroxidase-conjugated streptavidin (1:300 dilution). All steps were performed at room temperature. Tissue peroxidase activity was visualized by a 5-minute exposure to 3,3-diaminobenzidine tetrahydrochloride solution. Some sections were counterstained with hematoxylin.
Identification of Apoptotic Hepatocytes and Apoptotic Ballooned Hepatocytes
Apoptosis was identified by both light and electron microscopy if the cells exhibited any of the following morphological features: the formation of sharply delineated, uniformly finely granular masses or crescents; nuclear fragments; condensed cytoplasm; apoptotic bodies; and phagocytosis of apoptotic cells by adjacent cells.1-5 DNA fragmentation of apoptotic cells was identified by both positive staining from the TUNEL method and clearly visible nuclear fragments or sharp and condensed chromatic masses or crescents in the nuclei.
Cell Counting
We used the method described by Bouwens et al22 to quantify the cell number. For each rat, a total tissue area of approximately 8 mm2 (130 fields, 0.0625 mm2 per field) was counted from a random sample of six sections. The paraffin sections stained by the TUNEL method and the semithin (0.5-µm) sections stained with toluidine blue were evaluated by light microscopy at x250. The number of apoptotic hepatocytes that exhibited both TUNEL-positive staining and the clear, sharply condensed chromatic masses or crescents in nucleus or nuclear fragments were quantified. Only those ballooned cells with nuclei were counted in the semithin sections. The number of ballooned cells per mm2 was quantified. Those cells with condensed and crescent-shaped chromatic caps and/or nuclear fragmentation and apoptotic bodies were classified as apoptotic ballooned cells. The percentage of apoptotic ballooned hepatocytes in nucleated ballooned cells was analyzed by counting 400 nucleated cells in each rat.
Statistical Analysis
Data are expressed as mean ± SD. The significance of the difference between groups was evaluated by the two-tailed Student's t-test. Differences between treated groups and control groups were analyzed using the t-test for paired data. A level of P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ballooning of the hepatocytes with appearance of pale, foamlike
cytoplasm was first observed at the midzonal region of the lobule 3
hours after CCl4 administration (Figure 1A)
. The ballooned hepatocytes were
characterized by dilation of the endoplasmic reticulum (Figure 1B)
. At
this stage, apoptotic nuclei characterized by chromatin condensation
were found sporadically both in ballooned (Figure 1
, A and B) and
nonballooned hepatocytes (Figure 1C)
. Focal apoptosis was evident in
the lobule at 6 hours after CCl4 injection. Apoptotic
ballooned hepatocytes with nuclear fragments (Figure 1D)
and apoptotic
bodies (Figure 1E)
were frequently observed. By 6 to 12 hours after
CCl4 administration, individual necrotic cells were
observed in the intermediate area of the lobule (Figure 2A
, hepatocytes; Figure 2B
, ballooned
hepatocytes). Necrotic cells showed less electron density in both the
cytoplasm and the nucleus. The ballooned hepatocytes formed a circle
that surrounded the centrilobular zone of the lobule (Figure 2C)
. Some
nuclei of the ballooned cells were blebbing at this time (Figure 2D)
;
the blebs appeared to be shed into the dilated perinuclear space. The
heterochromatin also became condensed and margined. Fragments of the
condensed nuclei were often separated from the cells and phagocytosed
by adjacent cells (Figure 2E)
.
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, A and B). The
dilation of the cisternae in the ballooned hepatocytes progressively
increased until the cisternae occupied most of the cytoplasm (Figure 3C)
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The ballooned hepatocytes were first identified at 3 hours after
CCl4 injection; they increased rapidly in number, reaching
a maximum at 6 hours, and decreased thereafter. The percentage of
apoptotic ballooned cells continued to increase for up to 3 days after
the administration of CCl4 (Figure 4)
. Apoptotic bodies were also frequently
incorporated in the neighboring cells, such as ballooned hepatocytes
(Figure 5A)
, hepatocytes (Figure 5B)
,
Kupffer cells (Figure 5C)
, stellate cells (Figure 5D)
, and endothelial
cells.
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To quantify the apoptotic cells, low-molecular weight DNA was
extracted from the ethanol-fixed apoptotic cells, and this had
fractionated DNA content. The fluorescence of cells stained with
propidium iodide was measured with a FACS Calibur flow cytometer. The
number of apoptotic cells increased after the administration of
CCl4; most of these cells occurred in
sub-G0/G1 phase (Figure 6A)
, and their percentage peaked at 6
hours after the injection of CCl4. Treated groups showed
statistical significance compared with control (Figure 6B
;
P < 0.05). No ladder was observed in the control
cells; however, a ladder was observed at 6 hours after injection of
CCl4 (Figure 6C)
.
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As expected, few TUNEL-positive cells were observed in the liver
of the control rats. However, two types of dying cells were identified
in the experimental animals: TUNEL-positive cells with (type I; Figure 7
, A to C) and those without (type II;
Figure 7
, D and E) the classic morphological features of apoptosis (ie,
sharply delineated masses or crescents of condensed chromatin). Some
necrotic cells that were TUNEL negative exhibited a weakly and
nonspecifically stained nucleus and cytoplasm (Figure 7F)
.
TUNEL-positive apoptotic cells (type I cells) were first found
scattered in the lobules 3 hours after the administration of
CCl4. By 6 to 12 hours after the administration, the
TUNEL-positive cells were distributed in foci in the intermediate area
of the lobule, and by 24 to 48 hours, apoptotic foci were restricted to
the centrilobular area. The distribution of these cells coincided with
that of the phagocytes, which were also detected as Kupffer cells and
macrophages by immunoreaction with ED1 and ED2
Abs (Figure 7
, G and H). Double staining showed that the majority of
the apoptotic cells were in contact with, surrounded by, or situated
within, the ED1+ or
ED2+ Kupffer cells and/or macrophages that were
expressing MHC class II Ag labeled by OX-6 Ab (Figure 7
, I and J). The
number of TUNEL-positive apoptotic cells increased and peaked at 6
hours and then decreased rapidly from 48 hours after CCl4
administration (Figure 8)
. Numerous
Kupffer cells/macrophages incorporated the highly fragmented and dying
cells. This increase of apoptotic cells differed significantly from the
findings in the placebo-treated animals (P <
0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Physiologically, TUNEL positivity represents a criterion by which to identify apoptosis. Pathologically, a TUNEL-positive reaction can appear in both apoptosis and necrosis.20 Rink et al24 demonstrated two types of TUNEL-positive cells in brain injury by light and electron microscopy: one type displayed the morphological features of necrotic cell death, and the other type displayed the morphological features of classic apoptotic cell death. The former were interpreted as necrotic, and the latter apoptotic. A recent report mentioned that centrilobular hepatocytes were labeled by TUNEL staining after a single dose of CCl4.14 It remains to be determined whether the cells labeled by TUNEL staining are necrotic, whether apoptosis also occurs after CCl4, and whether it might contribute to liver injury regression. Data from the present study suggest that apoptosis, in addition to necrotic cell death, occurred after CCl4 administration. To make quantitative data of apoptosis more precise, it is necessary to differentiate between apoptosis and necrosis by combining TUNEL positivity and the nuclear morphology of dying cells.20 In response to the TUNEL staining, the TUNEL-positive cells were of two types. Type I cells, with TUNEL positivity of the nucleus and the accepted morphology, such as condensed and margined, crescent-shaped chromatic caps, were apoptotic. Type II cells, with features consistent with necrotic cell death, were necrotic. The manifestation of type I TUNEL-positive cells in the necrotic zone indicated that apoptosis represents one of the mechanisms of cell death after CCl4-induced liver injury.
The fate of the ballooned hepatocytes was not clearly established by
previous studies.16,17
Ballooned cells formed within 6
hours after a single dose of CCl4 and decreased in number
thereafter. Ballooned hepatocytes may undergo more profound hydrolytic
changes, such as necrosis (Figure 2B)
. In our model, however, there
were relatively few necrotic ballooned cells, whereas numerous
ballooned cells were undergoing apoptosis. Some of the ballooned
hepatocytes exhibited nucleoplasmic blebs that were shed into either
the dilated cisternae of endoplasmic reticulum or the perinuclear space
at the same time that the nuclei shrank and formed crescents. Although
nucleoplasmic blebbing may partially explain the nuclear pyknosis, the
mechanism underlying the blebbing phenomenon remains unknown. In
response to the elimination of the hepatocytes by apoptosis and
necrosis in the centrilobular zone, the cuff of ballooned cells (Figure 2C)
moved nearer the centrilobular veins. Based on a count of 400
ballooned cells, we found that, in contrast to the decrease in number
of ballooned cells per mm2, the percentage of apoptotic
ballooned cells increased up to 72 hours. The apoptosis of ballooned
cells is mainly responsible for their decrease and disappearance
(Figure 3D)
. Thus, it is tempting to consider that the ultimate fate of
the ballooned cell in this model was apoptosis.
Surprisingly, numerous hepatocytes in the centrilobular zone, the
so-called necrotic zone, were seen to undergo apoptosis in the 24 to
48-hour period after the administration of CCl4 (Figure 3
,
A and B). The quantification of apoptotic cells does suggest that the
high counts truly reflect a high cell loss, and that the injured cells
that constituted up to half the lobule had disappeared in response to
both apoptosis and necrosis. Furthermore, proliferating peripheral
cells had replaced this band of ballooned and centrilobular cells 72
hours after CCl4 administration. The lobule was completely
recovered by 4 days after the CCl4 injection.
The issue of the percentage of cells undergoing apoptosis and ballooning in vivo in CCl4-induced injury is more difficult to accurately calculate, because the loss of plasma membrane integrity, the hallmark of cell necrosis, made it impossible to estimate total hepatocytes (including necrotic cells). Instead, the numbers of TUNEL-positive cells and ballooned cells per mm2 are of significant value in this study. Regarding quantification of apoptotic ballooned cells among the total ballooned cells, it is obvious that the percentage of apoptotic ballooned cells is suitable, because there are few necrotic ballooned cells, and these can be neglected.
Apoptosis appears to be more advantageous than necrosis for removing injured hepatocytes. The latter leads to liberation of the proteins and DNA and to the formation of oxygen radicals, cytokines, and other inflammatory mediators, all of which may trigger potential secondary lethal responses within the organism. Extensive necrosis can damage the tissue structure and result in fibrosis. During the hepatic injury, apoptotic cells were found sporadically in the lobule at 3 hours after the administration of CCl4, and focal apoptosis was evident in the intermediate area at 6 to 12 hours and in the centrilobular area at 24 to 72 hours. Thus, the occurrence and distribution of apoptotic cells is shifted from that of acute injury to a resolution phase. Given that the duration of apoptosis in an individual cell is less than 3 hours,25 our results indicate that cell loss by apoptosis occurs fairly rapidly and that this can result in the shortening of the time course of the injury. It thus seems logical that a lack of apoptosis can contribute to a more persistent or chronic inflammatory response that, in turn, leads to fibrosis. This is especially true, given that a single injection of CCl4 does not induce fibrosis, whereas injections given twice a week for several weeks do so.26
The mechanisms for recognizing and eliminating apoptotic cells need to
be emphasized. Kupffer cells/macrophages, together with neighboring
hepatocytes, stellate cells, and endothelial cells, take part in
clearing out apoptotic hepatocytes, findings that agree with our
previous results.27
The majority of phagocytes stained
positively as both MHC class II (Ia) Ag+ and
ED1+ or ED2+ Kupffer
cells and macrophages. Because numerous apoptotic cells were surrounded
by Kupffer cells and macrophages (Figure 7
, G and I), it is suggested
that migration of the cells into the hepatic lobule is important for
recognizing and phagocytosing the apoptotic bodies.
Regarding the induction of necrosis or apoptosis by CCl4, one possibility is that there might be a dose dependency; CCl4 is capable of causing cell necrosis and will often induce apoptosis if the injury is milder and less severe. The dose of CCl4 that we used (0.15 ml/kg) is lower than that used by others.28 A second possibility is that apoptotic cells were digested by neighboring cells after tissue removal, in which case there would be an underestimation of the level of apoptosis; this cannot be ruled out, although the chance was minimized in this study, because the tissues were perfusion fixed. A third is that, unlike other organs, liver contains professional phagocytes: Kupffer cells, which normally represent 70 to 80% of the tissue macrophages and may accelerate elimination of apoptotic cells.
The mechanisms of drug-induced liver cell injury have recently been
reviewed,23,29
and several toxins described can damage DNA
by oxidation or alkylation, and ultimately lead to apoptotic cell
death. The events that trigger apoptosis in CCl4-induced
acute hepatic injury are as yet unknown. CCl4-induced
toxicity in isolated hepatocytes is mediated by a direct solvent injury
to cell membranes.30
However, nonparenchymal cells such as
Kupffer cells are activated by the release of cytokines, which may
contribute to pathophysiologic processes culminating in hepatocyte
apoptosis after toxic injury to the liver.28,31
We have
found that the concentration of apoptotic cells is highest in regions
enriched in macrophages, and it is possible that within these regions
macrophages are initiating and/or propagating these apoptotic events
(Figure 7
, H and J). Pretreatment with the Kupffer cell inhibitor
gadolinium chloride prevented cell death due to
CCl4.32
The induction of apoptosis by
macrophage is of interest, particularly considering recent reports that
apoptotic occurrence is consistent with macrophage cytocidal
activity,33-36
and that the aging
neutrophils37
and those cells from a regressing
tissue33-36
are engulfed before the death of the tissue
and subsequently acquire the morphology typical of apoptosis. Confirmed
mediators of macrophage cytotoxicity currently include tumor necrosis
factor 
, nitric oxide, and reactive oxygen
intermediates.36,38-42
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Acknowledgements |
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Footnotes |
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Supported by Grant C08457002 from the Japanese Ministry of Education, Science, Sports and Culture.
Accepted for publication May 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced apoptosis in hepatocytes in long-term culture. Am J Pathol 1996, 148:485-495[Abstract]
requires transcriptional arrest. J Immunol 1994, 153:1778-1787[Abstract]
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) reached a maximum
at 6 hours and then decreased. The percentage of apoptotic ballooned
hepatocytes (
) increased for up to 3 days after the
injection. Values are means ± SD. *P < 0.05 compared
with untreated controls.
, administered CCl4; 
, administered liquid paraffin.
Values are means ± SD. *P < 0.05 compared with
CCl4 (0
hours) or liquid paraffin treatment.