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Regular Articles |
in Liver Regeneration from Oval Cells
From the Department of Life Sciences and Chemistry,*
Roskilde University, Roskilde, Denmark; the First Institute of
Pathology and Experimental Cancer Research,
Semmelweis Medical University, Budapest, Hungary; and the Laboratory of
Experimental Carcinogenesis,
Division of
Basic Sciences, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor 
subunit (IFN-R),
gp91phox, interleukin-1ß (IL-1ß), lymphocyte
function-associated molecule-1 (LFA-1), eukaryotic
initiation factor-2-associated 67-kd protein (eIF-2-associated 67-kd
protein), and -fetoprotein, which constitute part of
the cellular program modulated by IFN-. Therefore,
expression analysis performed by Northern blotting and
immunohistochemistry were extended to include IFN-, the
IFN- receptor ß subunit (IFN-Rß), three secondary
response genes induced by interaction of IFN- with IFN- receptor
complexes, ie, IL-1ß-converting enzyme (ICE),
intercellular adhesion molecule-1 (ICAM-1), and urokinase-type
plasminogen activator receptor (uPAR), and a cytokine inducing
IFN- expression, ie, interleukin-18 (IL-18). The
Northern blot analysis showed that all examined genes were modulated
when progenitor-like oval cells were activated and recruited for liver
regeneration. Immunohistochemistry localized the subunits of the
IFN- receptor complex, IFN-R and IFN-Rß,
the secondary response genes uPAR and ICAM-1, the
IFN--inducing factor IL-18, and ICE to the ductular
structures of oval cells. In contrast, during liver
regeneration after a 70% partial hepatectomy, only modulation
of IL-1ß and ICE was observed. Our results,
therefore, indicate that IFN--mediated events may be
particularly important when cells in the bile ductules must respond to
liver damage by production of ductular oval cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several experimental rodent models have been used to study oval cell
proliferation and differentiation in vivo.5
We
have focused our efforts on studying the mechanisms involved in oval
cell activation, proliferation, and differentiation in the rat liver
using the modified Solt-Farber protocol, in which feeding of
2-acetylaminofluorene (2-AAF) is combined with a 70% partial
hepatectomy (AAF/PHx). Feeding with 2-AAF activates cells in the bile
ductules to undergo replication and acquire an oval cell phenotype with
expression of hepatocyte specific genes, and the partial hepatectomy
provides the necessary stimulus to induce massive proliferation and
migration of the activated oval cells into the liver parenchyma, where
they differentiate to give rise to mature
hepatocytes.2,6,7
Our studies have indicated the
involvement of a number of growth modulators, including stem cell
factor (SCF), transforming growth factor (TGF-), epidermal
growth factor (EGF), hepatocyte growth factor (HGF), urokinase-type
plasminogen activator (uPA), leukemia inhibitory factor (LIF), and
their receptors in oval cell proliferation, and the cellular
localization of the different components indicates that an intricate
network of paracrine and autocrine mechanisms of growth modulation is
involved.8-13
Despite the research efforts, our knowledge of the mechanisms involved in oval cell activation and differentiation is still limited. To acquire a better understanding of the mechanisms involved, we have begun identifying and characterizing genes that are highly expressed when oval cells are activated and recruited for liver regeneration. In the present study, we have used suppression subtractive hybridization to clone genes with increased expression during oval cell proliferation.
We report here the identification of several genes that we believe may
play a role in generating the oval cell response in vivo. A
common feature of the identified genes is their connection to the
cellular programs orchestrated by interferon- (IFN-). Using
Northern blot and immunohistochemical studies, we further investigated
the expression of a number of genes connected to the modulating network
of IFN-. We show that these genes, which include IFN- itself, the
IFN- receptor subunits (IFN-R) and ß (IFN-Rß),
primary and secondary response genes induced by interaction of IFN-
with IFN- receptor complexes (ie, gp91phox,
interleukin-1ß-converting enzyme (ICE), intercellular adhesion
molecule-1 (ICAM-1), and urokinase-type plasminogen activator receptor
(uPAR)), cytokines inducing IFN- expression (ie, interleukin-18
(IL-18) and interleukin-1ß (IL-1ß)), and cell adhesion molecules
orchestrating lymphocyte-epithelial cell interactions (ie, lymphocyte
function-associated molecule-1 (LFA-1)) are all modulated when
progenitor-like oval cells are activated and recruited for liver
regeneration in the AAF/PHx protocol. However, during liver
regeneration from existing mature liver cells, modulation of the
majority of these genes cannot be observed. Therefore, our results
indicate that IFN--mediated events may be particularly important
when cells in the bile ductules are activated in response to liver
damage by production of ductular oval cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In experiments combining the feeding of 2-AAF with a two-thirds partial hepatectomy (AAF/PHx), 8- to 9-week-old male Fischer 344 rats (120–150 g body weight) were given a daily dose of 20 mg/kg 2-AAF by gavage for 4 consecutive days. On day 5 after initiation of the 2-AAF treatment, resection of approximately 70% of the liver (left lateral and median lobe PHx) was performed, followed by administration of 2-AAF for an additional 4 days. All animals were kept under standardized conditions with access to food and water ad libitum and maintained according to the guidelines established at the National Institutes of Health.
For Northern blot analysis, animals were sacrificed 96 hours after initiation of 2-AAF treatment and 7, 8, 10, and 11 days after PHx was performed. After excision of the liver, sections of all liver lobes were fixed in formalin, and the remaining liver tissue snap-frozen in liquid nitrogen. Liver tissue for Northern blot analysis was also obtained from animals sacrificed 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, or 60 hours after a 70% hepatectomy (PHx) without administration of 2-AAF. For isolation of nonparenchymal cell populations after exposure to 2-AAF, animals were treated according to the AAF/PHx protocol or received a daily dose of 20 mg/kg 2-AAF by gavage for up to 96 hours and used for cell isolation as described below.
Isolation of Nonparenchymal Liver Cells and Construction of the Suppression Subtractive Hybridization Library
Although a number of cell types comprised of parenchymal
hepatocytes as well as nonparenchymal bile duct, stellate, endothelial,
and Kupffer cells may be involved in generating the oval cell response,
we were particularly interested in identifying genes that were
differentially expressed in the nonparenchymal cell compartments in the
liver regenerating from oval cells. Because our previous studies in the
AAF/PHx protocol have shown the presence of numerous ductular
structures of proliferating oval cells and no overt differentiation of
oval cells into foci of basophilic hepatocytes and intestinal-type
structures at day 7 after PHx,2,7
nonparenchymal cell
populations containing oval cells were separated from hepatocytes by
perfusion of rat livers at this time point (AAF/PHx 7d) and, together
with nonparenchymal cell populations isolated from normal liver
(control), used for generation of the suppression subtractive
hybridization library. The isolation procedure has been described in
detail by Bisgaard et al.11
In brief, liver cells were
released by a three-step perfusion procedure in situ,
employing first a perfusion with Hanks' balanced salt solution without
calcium and magnesium, second a perfusion with 0.2% pronase, and third
a perfusion with 0.1% pronase, 0.1% collagenase type I, and 0.007%
DNase I. Viable nonparenchymal cell populations were purified by
centrifugation through a two-step Percoll gradient. To ensure that the
isolated nonparenchymal cells were comprised of the expected cell
populations represented by oval, bile duct, stellate, Kupffer, and
endothelial cells, we analyzed the expression of a number of factors
expected to be differentially expressed between AAF/PHx cells and cells
isolated from control liver. The parameters included
-glutamyltranspeptidase (-GT) as a marker for epithelial cells of
ductal origin, -fetoprotein14
as a marker for oval
cells, and HGF10
and uPA11
as markers
for cells of nonepithelial origin. In the nonparenchymal cell
populations from control liver approximately 10 to 20% of the cells
stained positive for -GT, indicating their ductal origin. In
accordance with the increased numbers of -GT-positive cells observed
in livers regenerating by recruitment of oval cells,13
30
to 50% of the cells isolated from AAF/PHx 7d livers were positive for
-GT. Other genes expressed in nonparenchymal cell populations during
liver regeneration from oval cells, including -fetoprotein, HGF, and
uPA, were all increased in nonparenchymal cell populations from
AAF/PHx-treated animals relative to cell populations obtained from
control liver when assessed by Northern blot analysis (data not shown).
Thus, the isolated nonparenchymal populations were comprised of the
expected cell populations and were subsequently used in the suppression
subtractive hybridization analysis. The final nonparenchymal cell
preparations contained less than 1% hepatocytes.
Total RNA from isolated nonparenchymal cell preparations was obtained by lysis of cell pellets in RNAstat Reagent (Tel-Test Inc., Friendswood, TX) and polyadenylated RNA (poly(A)+) selected by passage over an oligo(dT) column (Invitrogen Corp., Carlsbad, CA). The suppression subtractive hybridization was performed with the Clontech PCR-Select cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer's instructions. Samples of 2-µg poly(A)+ RNA were reverse-transcribed to cDNA. In the present study, we were interested in identifying genes that were modulated in nonparenchymal cells from rat liver regenerating after the combined feeding of 2-AAF and a PHx (AAF/PHx 7d mRNA) as compared to nonparenchymal cells isolated from normal rat liver (control mRNA). The cDNA synthesized from AAF/PHx 7d mRNA was therefore used as tester cDNA and cDNA generated from control mRNA as driver cDNA. In brief, the tester and driver cDNA were digested with RsaI and the tester cDNA ligated to the adapter DNA. After two repeated hybridizations of tester and driver cDNA, remaining unhybridized sequences (representing cDNAs expressed highly in the tester sample) were amplified by polymerase chain reaction (PCR) using flanking and nested primers that anneal the adaptor cDNA and the subtracted PCR products ligated into the pCR 2.1 plasmid vector (TA Cloning kit, Invitrogen). The identity of the cDNA inserts was revealed by sequence analysis of purified plasmids using the Thermo Sequenase Cycle Sequencing Kit (Amersham Life Science, Cleveland, OH), and comparison to known DNA sequences using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/).
Isolation of Nonparenchymal Cells for Northern Blot Analysis
Cell populations enriched in bile epithelial and/or oval cells were isolated according to the protocol described by Bisgaard et al.11 Animals treated with 2-AAF were sacrificed 0 (normal), 3, 6, 24, 48, and 96 hours after initiation of the 2-AAF treatment regimen. Nonparenchymal cell populations were isolated by perfusion and Percoll purification as described above. Kupffer cells were removed from the nonparenchymal cell populations by selective adherence to plastic tissue culture dishes. Removal of macrophages, endothelial cells, and red blood cells was achieved by selective panning using the mouse monoclonal antibody OX43 (MCA276; Serotec, Accurate Chemical and Scientific Corp., Westbury, NY). Cell preparations were snap-frozen in liquid nitrogen and stored at -70°C until processed for total RNA isolation and Northern blot analysis.
Northern Blot Analysis
Polyadenylated RNA was prepared from snap-frozen liver tissue by
ultracentrifugation through a cesium chloride cushion and enrichment by
oligo(dT)-cellulose chromatography. Samples of 5 µg
poly(A)+ RNA or 20 µg total RNA were
electrophoresed in 1% agarose/0.2 formaldehyde gels, transferred onto
nylon membranes, and hybridized to cDNA probes labeled with
[32P]dCTP (rediprime,
Amersham Life Science) using the QuickHyb solution from Stratagene (La
Jolla, CA). The following rat cDNAs were obtained from the subtraction
analysis: IFN-R (homology to mouse IFN-R, accession number
M28995, 85% identity); gp91phox (homology to mouse gp91phox, accession
number U43384, 96% identity); IL-1ß (homology to rat IL-1ß,
accession number M98820, 98% identity); eIF-2-associated 67-kd protein
(homology to rat eIF-2-associated 67-kd protein, accession number
L10652, 100% identity); LFA-1 (homology to mouse LFA-1, accession
number M60778, 91% idenitity); -fetoprotein (homology to rat
-fetoprotein, accession number X02361, 98% identity); ebnerin
(homology to rat ebnerin, accession number U32681, 100% identity). A
cDNA fragment of mouse uPA (kindly provided by Dr. J. L. Degen,
Children's Hospital Medical Center, Cincinnati, OH) and rat uPAR
(kindly provided by Dr. D. Dichek, J. David Gladstone Institutes, San
Fransisco, CA) were also used as probes.
The cDNA probes for IFN-, IL-18, ICE, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were generated by reverse transcription and
subsequent amplification by polymerase chain reaction (RT-PCR).
Poly(A)+ RNA (0.5 µg) isolated from whole liver
homogenate at day 10 in the AAF/PHx protocol (AAF/PHx 10d) was reverse
transcribed using MMLV-Reverse transcriptase and
oligo(dT)18 primers (Advantage RT-for-PCR kit,
Clontech). The cDNAs provided templates for PCR using the following
forward and reverse primers: rat IFN- (404 bp),
5'-tactgccacggcacagtcattgaa-3' and 5'-gcagcgactccttttccgcttcct-3'; rat
IL-18 (446 bp), 5'-actgtacaaccgcagtaatacgg-3' and
5'-agtgaacattacagatttatccc-3'; rat ICE (1189 bp),
5'-atggccgacaaggtcctgagggc-3' and 5'-agaaacgttttgtcagggtca-3'; and rat
GAPDH (651 bp), 5'-accacagtccatgccatcac-3' and
5'-tccaccaccctgttgctgta-3'. Oligonucleotide primers were derived from
published sequences in the GenBank/EMBL/DDBJ databank. All RT-PCR
products were ligated into the pCRII plasmid vector (TA Cloning kit
Dual Promoter, Invitrogen) and sequenced to confirm their identity.
Immunohistochemistry
Livers were fixed in 10% neutral formalin and processed for routine histology. Immunohistochemistry was performed on 5-µm tissue sections as previously descibed.11 Specific antibody binding was revealed by the appropriate Vectastain ABC Elite kits with diaminobenzidine as substrate in combination with an enhancing solution (Vector Laboratories, Burlingame, CA) followed by light counterstaining with Gill's hematoxylin.
The primary antibodies were as follows: a goat polyclonal antibody
against the carboxy terminus of the IL-18 precursor of mouse origin
(diluted 1:100); a rabbit polyclonal antibody against the carboxy
terminus of the p20 subunit of mouse ICE (diluted 1:500); a rabbit
polyclonal antibody raised against the carboxy terminus of the mouse
interleukin-1 receptor I (IL-1RI) precursor (1:25); a rabbit
polyclonal antibody directed against the carboxy terminus of
IFN-R (diluted 1:500); a rabbit polyclonal antibody against the
carboxy terminus of the IFN-Rß precursor of mouse origin (diluted
1:250); and a goat polyclonal antibody raised against the carboxy
terminus of mouse ICAM-1 (1:1000). These antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal
antibody against rat uPAR (diluted 1:10) was obtained from American
Diagnostica (Greenwich, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we used the suppression subtractive
hybridization technique to clone the genes expressed in nonparenchymal
cell populations during activation and proliferation of oval cells in
the AAF/PHx protocol. A subset of the identified genes encoded
components of the gene network connected to the proinflammatory
cytokine IFN-, ie, IFN-R, gp91phox, IL-1ß, LFA-1,
eIF-2-associated 67-kd protein, and -fetoprotein (Table 1)
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-fetoprotein,
albumin, and hepatic transcription factors within 96 hours after
exposure to the chemical.6
The subsequent partial
hepatectomy then provides the necessary growth stimulus for the massive
proliferation, expansion, and finally differentiation of the
AAF-induced oval cells in the liver parenchyma.2,7
To
investigate if the genes cloned by suppression subtractive
hybridization were modulated when oval cells were activated and
recruited for liver regeneration, we performed a Northern blot analysis
with whole liver mRNA extracts derived from a time course experiment
with the AAF/PHx protocol. The time points of the experiment were
chosen to investigate the modulation of the identified genes during i)
the induction of the oval cell phenotype in response to 2-AAF, which
occurs within 96 hours after initiation of the 2-AAF
exposure,6
ii) the expansion of the oval cell populations
into the liver parenchyma which can be observed at PHx 7d and
8d,2,7
and iii) differentiation of oval cells resulting in
development of foci containing basophilic hepatocytes, which can be
observed at PHx 10d and 11d.2,7
The Northern blot analysis
revealed that the steady-state mRNA levels of the cloned genes
increased in a manner consistent with activation of oval cells by 2-AAF
(Figure 1)
. However, whereas transcripts
for IFN-R, gp91phox, IL-1ß, LFA-1, and eIF-2-associated 67-kd
protein were detected at low levels in normal liver and increased in
response to treatment with 2-AAF, -fetoprotein and ebnerin mRNA were
detected in livers only after treatment with 2-AAF. In the present
study, high steady-state levels of transcripts for all genes were
detected 10 and 11 days after partial hepatectomy, a time when the
number of oval cells peaked, as revealed by morphological studies (data
not shown). Although the analysis of GAPDH expression was intended to
provide an internal control for the amount of mRNA loaded, the
expression of the gene was increased during liver regeneration in the
AAF/PHx protocol (Figure 1)
. Similar results were obtained for
ß-actin, which excluded a densitometric analysis as an appropriate
numerical basis for comparison of the Northern blots. However, the
GAPDH expression did confirm the integrity of the isolated mRNA.
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As demonstrated in Figure 1
, the steady-state mRNA levels of the
studied genes were increased in the liver within 96 hours after
treatment with 2-AAF had begun. However, the increased expression could
be due to an increased number of cells expressing a certain gene or,
alternatively, to an induction of gene expression in certain cell
populations. To evaluate the modulation of the cloned genes in response
to 2-AAF with special emphasis on the modulation in various cell
populations in the liver, we next separated the cells from rat livers
exposed to 2-AAF for up to 96 hours by selective perfusion techniques
and extracted total RNA. The separated cell populations consisted of
either purified hepatocytes or nonparenchymal cells devoid of Kupffer
cells, macrophages, endothelial cells, and red blood cells. Northern
blot analysis showed that although none of the genes except GAPDH was
expressed at significant levels in hepatocytes (data not shown), they
were expressed in cell populations enriched in bile epithelial and/or
oval cells (Figure 2)
. gp91phox, IL-1ß,
-fetoprotein, and ebnerin showed low expression in samples from
normal rats but were induced within 96 hours after exposure to 2-AAF.
In contrast, nonparenchymal cell populations isolated from normal rats
showed high steady-state levels of IFN-R and eIF-2-associated
67-kd protein, the expression of which did not change significantly in
response to 2-AAF. Therefore, we have identified at least two
categories of genes by suppression subtractive hybridization. One
category represents genes with induced expression in response to 2-AAF.
The other category of genes is constitutively expressed in a specific
cell type, but the relative abundance of the cells in the
nonparenchymal cell populations isolated at day 7 in the AAF/PHx
protocol may be increased as compared to nonparenchymal populations
isolated from control liver.
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-fetoprotein has
previously been shown to be a feature of activated bile ductular cells
and expanding oval cells in the AAF/PHx protocol.6,14
Ebnerin expression has not been reported in oval cells, but a
homologous gene has been shown to be expressed in the bile ducts of the
adult mouse liver.15
Therefore, we conclude that the
suppression subtractive hybridization technique has provided us with a
number of genes that are modulated when cells in the ductular
structures are activated to acquire an oval cell phenotype in response
to 2-AAF and stimulated to give rise to expanding populations of
ductular oval cells in the AAF/PHx protocol of liver regeneration.
Of known oval cell markers, only -fetoprotein was identified in our
initial screen of the suppression subtractive hybridization library.
However, other genes expressed in oval cells, such as the embryonic
form of glutathione-S-transferase (Yp form) and
-GT, would also be expected to be represented in the library. At the
present time we have sequenced 50 clones containing cDNA fragments but
are currently expanding our analysis of the library. We expect that the
increased number of clones analyzed will result in detection of other
known oval cell markers and identify genes which, as shown for ebnerin,
may be induced in nonparenchymal liver cells in response to 2-AAF.
IFN-, IGIF/IL-18 and ICE Are Expressed during Liver Regeneration
from Oval Cells
Since the subtraction suppression hybridization had identified
several genes expressed as components of the cellular programs
orchestrated by IFN- (Table 1)
, we investigated the expression of
IFN- in the AAF/PHx protocol of liver regeneration. As shown by the
Northern blot analysis in Figure 3
,
IFN- steady-state mRNA levels were not detectable in normal liver,
but increased to low but detectable levels during oval cell activation
and proliferation. Having established that IFN- expression is
induced in the AAF/PHx protocol, we then wanted to study the expression
of genes involved in IFN- production (ie, IL-18 and ICE). As
demonstrated in Figure 3
, IL-18 transcripts were detected at very low
levels in normal rat liver. Treatment with 2-AAF resulted in increased
steady-state levels of IL-18 transcripts, and, as oval cells expanded
into the parenchyma in response to the PHx, the levels increased
further and remained elevated throughout the time frame of the
experiment. Immunohistochemistry on tissue sections at day 7 after PHx
showed that IL-18 protein was highly expressed in sinusoidal lining
cells as well as in the ductular structures of oval cells (Figure 4B)
. Hepatocytes did not show significant
staining.
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production.16
However, in hepatocytes IL-1ß has also been shown to induce IFN-
production.17
Having demonstrated that expression of both
IL-1ß and IL-18 is induced in populations containing oval cells, we
next wanted to examine the expression of ICE, the protease that
converts the precursors of the two cytokines to their active
forms.18,19
Low steady-state mRNA levels of ICE could be
detected in normal liver, but the levels were highly elevated after
treatment with 2-AAF and increased further as oval cells proliferated
and expanded into the liver parenchyma (Figure 3)
. Immunohistochemistry
for ICE protein showed strong staining over the ductular structures of
oval cells (Figure 4C)
. Therefore, proliferating oval cells appeared
able to produce the precursors of IL-ß and IL-18 and to process the
proteins to their active forms. Strong immunohistochemical staining for
IL-1RI, the high-affinity receptor for IL-1ß, was detected in
oval cells as well as in the hepatocytes (Figure 4D)
, suggesting that
IL-1ß may be acting by both autocrine and paracrine mechanisms during
liver regeneration in the AAF/PHx protocol.
The Receptors for IFN- Are Highly Expressed in Ductular
Structures of Oval Cells
IFN- interacts with a specific cell surface receptor consisting
of two subunits: the -chain, exhibiting high-affinity ligand binding
properties, and the ß-chain, required primarily for
signaling.20
In the AAF/PHx protocol, IFN-R protein
was detected in ductular structures of oval cells as well as in cells
located in the sinusoids (Figure 4E)
. The protein was not detected in
mature hepatocytes.
Ductular structures of oval cells also showed a strong positive
reaction for IFN-Rß protein, whereas mature hepatocytes stained
weakly positive (Figure 4F)
. Unlike IFN-R, IFN-Rß was not
detected in other nonparenchymal cell populations in the liver.
Therefore, in the AAF/PHx protocol, the ductular structures of oval
cells express the highest levels of the IFN- receptor subunits,
which are both required for proper cellular signaling by IFN-.
Genes Regulated in Response to IFN- Signaling Are Induced in
Oval Cells
A number of genes are regulated in response to ligation of IFN-
to its receptor.20
gp91phox, which is induced as a primary
response gene, was cloned in the present study by suppression
subtractive hybridization (Figure 1)
and shown to be induced in
nonparenchymal cell populations containing activated bile duct and/or
oval cells (Figure 2)
. Furthermore, other genes induced by IFN-,
including the protease uPA and its receptor uPAR, were also induced in
these cell populations as revealed by Northern blot analysis and
immunohistochemistry (Figures 3 and 4G)
, as was ICAM-1, which is
characterized as a secondary response gene to IFN- (Figure 4H)
.
These observations support the hypothesis that IFN- is an important
player in the establishment of the oval cell phenotype in the AAF/PHx
protocol.
Expression of Genes in the IFN- Modulating Network during Liver
Regeneration from Existing Mature Epithelial Cells (PHx)
In recent years it has become apparent that many of the numerous
growth factors and cytokines, notably HGF, TGF-, EGF, tumor necrosis
factor (TNF-), and IL-6, that initiate or facilitate the normal
regenerative response from existing liver cells (ie, hepatocytes and
bile duct cells) after PHx are also implicated in regeneration from
oval cells.1,3
An increase in HGF mRNA is observed in
hepatic stellate cells 3 to 6 hours after a 70% PHx and gradually
returns to undetectable levels within 72 to 96 hours after
PHx.21
Similarly, TGF- mRNA is induced in hepatocytes
within 2 to 3 hours after PHx, rises to a peak between 12 and 24 hours,
and remains elevated for at least 48 hours after PHx.22
In
the AAF/PHx protocol of liver regeneration, HGF and TGF- mRNA are
induced 96 hours after exposure to 2-AAF, are further increased in
response the PHx, and remain expressed at high levels throughout the
period of oval cell expansion and differentiation, to return to levels
seen in normal liver at the end of the regeneration
process.23
These observations prompted us to investigate
if the IFN- modulating network was also involved in regeneration
after a 70% PHx. A Northern blot analysis spanning the first 60 hours
after PHx showed that only IL-1ß mRNA reached steady-state levels
comparable to those observed in the AAF/PHx protocol of liver
regeneration (Figure 5)
. Interleukin-1ß
transcripts were detected in two major peaks 12 and 36 hours after PHx.
Low steady-state levels of transcripts for IFN-R, IL-18, and ICE
were detectable in all samples when probe hybridizations were performed
in parallel with the AAF/PHx experiments and exposure times of the
autoradiographs were identical. A small elevation in transcript levels
over control for IL-18 and ICE was observed 42 hours after PHx, when
DNA synthesis in the bile duct epithelium is at its
maximum.1
Transcripts for IFN-, LFA-1, -fetoprotein,
and ebnerin were undetectable (data not shown). Therefore, the
IFN--modulating network appears to be a major player in the
regenerative process only when oval cells are activated and recruited
for tissue repair as exemplified by the AAF/PHx model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a pleiotropic cytokine orchestrating a
remarkable range of distinct cellular programs.20
The
effects are implemented by complex patterns of cell-specific gene
regulation mediated by binding of the cytokine to IFN- receptors on
the cell surface, followed by activation of intracellular signaling
cascades and activation of both primary and secondary response genes.
The IFN- response is itself regulated by other cytokines including
IL-18 and IL-1ß.16,17
The present study has for the
first time demonstrated that genes that are integral parts of the
cellular programs activated by IFN- are modulated when oval cells
are activated and recruited for tissue repair in the AAF/PHx protocol.
The identified genes include IFN- itself, the receptors for IFN-
(IFN-R and IFN-Rß), primary (gp91phox) and secondary
response genes (ICE, ICAM-1 and uPAR), cytokines inducing IFN-
expression (IL-18 and IL-1ß), and, finally, cell adhesion molecules
orchestrating lymphocyte-epithelial cell interactions (LFA-1 and
ICAM-1; Table 1
).
We have shown that ductular structures of oval cells in the
regenerating liver express the and ß subunits of the IFN-
receptor complex. Because both subunits are required for proper
signaling on ligation of IFN-, these cell populations may be primary
targets of IFN- when cells in the bile epithelial structures are
activated to generate proliferating ductular structures of oval cells.
In the AAF/PHx model of liver regeneration, the oval cells later
differentiate to give rise not only to new hepatocytes but also to
intestinal-like structures.2,3
Evidence supporting
a role for IFN- as a major player in ductal cell proliferation and
tissue regeneration has been obtained from studies in transgenic mice
engineered to constitutively express the mouse IFN- gene in either
hepatocytes, under control of the human serum amyloid P component gene
promoter, or in pancreatic islet cells driven by the human insulin gene
promoter.24,25
Hepatocyte-specific production of IFN-
was sufficient to induce chronic inflammatory disease with persistent
liver cell damage and proliferation of cells in ductular
structures.24
Similarly, in the pancreas, expression of
the cytokine in islet cells elicited destruction of islets and, to some
extent, of acinar tissue.25
However, IFN- expression in
islet cells also initiated an islet regeneration process evidenced by a
flurry of mitotic activity in pancreatic duct cells and the appearance
of three major endocrine cells in the duct walls and in the islet-like
cell aggregates. Additionally, some pancreatic duct cells expressed
albumin and -fetoprotein and occasionally differentiated into
hepatocytes and intestinal-like cell types. In summary, the studies
performed with transgenic mouse models expressing IFN- in both liver
and pancreas underscore the importance of this cytokine in the
mitogenic activation of cells in the hepatic and the pancreatic ducts
and the cytokine's ability to modulate cellular differentiation
pathways, at least in the pancreas.
We have established that IFN- and several of the genes it regulates
through ligation to the IFN- receptor complex are modulated in cell
populations responding on exposure to 2-AAF. However, the mechanisms by
which IFN- may activate cells in bile ductules to enter DNA
synthesis and divide, and whether the same mechanisms are involved when
progeny of oval cells migrates and expand into the liver parenchyma,
are not known. It is well recognized that cytokines modulating the
immune system such as IFN- act in synergy with growth factors in
complex networks to regulate biological processes. Recently, in
vitro studies have shown that IFN- by itself has very little
effect on cellular DNA synthesis. In contrast, the cytokine
dramatically enhances DNA synthesis in response to mitogenic growth
factors such as EGF.26
The interaction between IFN- and
EGF appears to be mediated by signal transducer and activator of
transcription 1 (STAT1), a member of the STAT family of
transcription factors. Because a number of mitogenic growth
factors including TGF-, EGF, and HGF has been shown to be involved
when oval cells proliferate in the AAF/PHx
protocol,9,10,13
it is possible that IFN- may prime
ductal cells in vivo through activation of signal
transducers that may augment the mitogenic response of the cells to
other cytokines and growth factors.
We have previously reported that cells in the bile ductules enter DNA
synthesis after exposure to 2-AAF.6
In the present study
we have shown that expression of uPAR, which is induced as a secondary
response gene to IFN-, is already up-regulated 96 hours after
exposure to 2-AAF and is expressed in the infiltrating ductular
structures of oval cells. As an essential component in the plasminogen
activator/plasmin system uPAR acts as the cellular receptor for uPA,
and formation of the receptor-ligand complex results in proteolytic
activation of plasminogen to plasmin on the surface of activated
ductular and/or oval cells.11
The plasminogen
activator/plasmin cascade is involved in fibronolysis and degradation
of extracellular matrix (ECM) components, but has also been implicated
in activation of latent growth factors.27
One of these
factors, the heparin-binding glycoprotein HGF, a powerful mitogen for
liver epithelial cells in vitro and considered to play a
major role in liver regeneration in the AAF/PHx
protocol,10
is converted to its biologically active form
by the uPA/uPAR complex.28
Therefore, we find it
reasonable to hypothesize that another priming effect of IFN- is to
induce expression of uPAR on the surface of bile ductular cells. After
binding of uPA, the uPAR/uPA complex will mediate generation of plasmin
in the immediate vicinity of the ductular structures, degradation of
the surrounding extracellular matrix, and localized liberation and
activation of HGF. Mitosis may then be initiated by a paracrine
interaction of activated HGF with its receptor on the ductular cells.
Supporting this hypothesis is our recent demonstration that both uPA
and HGF enhance the mitogenic response in the bile duct epithelium on
infusion in 2-AAF treated animals.13
As discussed above, a number of the cellular responses of hepatic
ductular cells to 2-AAF may be orchestrated by ligation of IFN- to
its cell surface receptor complex. However, IFN- production is
itself subject to complex regulatory pathways. One of the factors
involved in IFN- production is IL-18.29
The cytokine is
synthesized as a leaderless precursor requiring ICE for cleavage into
the active molecule. In the present study, we have demonstrated that
the steady-state levels of IL-18 and ICE transcripts increase when oval
cells are recruited for liver regeneration in the AAF/PHx protocol.
Furthermore, we have demonstrated the presence of both IL-18 and ICE
protein in the ductular structures of oval cells. Taken together, these
observations support an important function of IL-18 in activation and
expansion of oval cell populations, possibly as a primary mediator of
IFN- production.
In summary, using suppression subtractive hybridization, Northern blot,
and immunohistochemical analyses in the AAF/PHx model of liver
regeneration, we have identified several genes that are modulated when
oval cells are activated in response to the liver damage inflicted. A
common feature of the identified genes is their connection to the
cellular programs orchestrated by IFN-, indicating an important role
for IFN- as a growth-modulating molecule when epithelial cells in
the bile ductules are activated and induced to produce a large
population of oval cells.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Danish Cancer Society (to H. C. B. and L. J. R.), the Novo Nordisk Foundation (to H. C. B.), the Danish Natural Science Research Council (to H. C. B.) and the Danish Medical Research Council (to L. J. R.). P. N. was supported by a NATO linkage grant.
Accepted for publication June 16, 1999.
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References |
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