1 Boehringer Ingelheim Research
Group, and 2 First Department of Medicine, Transforming
growth factor-
transforming growth factor- HEPATIC FIBROSIS is a dynamic process caused by chronic
liver injury due to various etiologies (viral, toxic, metabolic,
autoimmune), eventually leading to cirrhosis. It is predominantly
characterized by excessive accumulation of extracellular matrix caused
by both an increased synthesis and decreased or unbalanced degradation of extracellular matrix. Transforming growth factor (TGF)- Importantly, animal models of experimental liver fibrosis, such as
carbon tetrachloride-induced fibrosis (8), bile duct ligation (39), or
schistosomiasis infection (17), support the in vivo relevance of
TGF- To develop a transgenic mouse model with highly liver-specific,
inducible expression of TGF- Construction of the TGF-
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (TGF-
1) is a powerful stimulus for collagen
formation in vitro. To determine the in vivo effects of TGF-
1 on
liver fibrogenesis, we generated transgenic mice overexpressing a
fusion gene [C-reactive protein (CRP)/TGF-
1] consisting
of the cDNA coding for an activated form of TGF-
1 under the control
of the regulatory elements of the inducible human CRP gene promoter.
Two transgenic lines were generated with liver-specific overexpression
of mature TGF-
1. After induction of the acute phase response (15 h)
with lipopolysaccharide (100 µg ip), plasma TGF-
1 levels reached
>600 ng/ml in transgenic animals, which is >100 times above normal
plasma levels. Basal plasma levels of uninduced transgenic animals were
about two to five times above normal. As a consequence of hepatic
TGF-
1 expression, we could demonstrate marked transient upregulation
of procollagen I and procollagen III mRNA in the liver 15 h after the
peak of TGF-
1 expression. Liver histology after repeated induction
of transgene expression showed an activation of hepatic stellate cells
in both transgenic lines. The fibrotic process was characterized by
perisinusoidal deposition of collagen in a linear pattern. This
transgenic mouse model gives in vivo evidence for the important role of
TGF-
1 in stellate cell activation and liver fibrogenesis. Due to the
ability to control the level of TGF-
1 expression, this model allows
the study of the regulation and kinetics of collagen synthesis and
fibrolysis as well as the degree of reversibility of liver fibrosis.
The CRP/TGF-
1 transgenic mouse model may finally serve as a model
for the testing of antifibrogenic agents.
1; stellate cell activation; collagen
I; C-reactive protein promoter
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 seems to
play a central role in the cytokine network involved in fibrogenesis (for review, see Refs. 5, 7, 21, 32, 48). High levels of TGF-
1 have
been described in different acute and chronic liver diseases (4,
11, 40, 49). TGF-
1 is the prototype and best characterized of
three TGF-
isoforms (TGF-
1, TGF-
2, TGF-
3) encountered in
mammalian species. It is a highly conserved molecule with a variety of
signalling functions regulating cell growth, differentiation,
migration, death, and expression of extracellular matrix (for review,
see Refs. 31, 47, 62). TGF-
1 is secreted in a biologically inactive
form as a complex consisting of two units of the large precursor
segment of the TGF-
1 propeptide linked to the mature TGF-
1 dimer.
In its biologically active form, TGF-
1 consists of a 25-kDa
homodimer linked by disulfide bonds. Conversion of the latent complex
to the mature, biologically active form is achieved in vitro by acid,
alkali, heat, or proteases (24, 35). In vivo mechanisms of TGF-
1
activation are not fully understood but seem to involve proteases (1,
54). In hepatic fibrogenesis, TGF-
1 is believed to be involved in
the synthesis and deposition of extracellular matrix components like fibronectin, collagens type I, III, and IV, tenascin, elastin, osteonectin, biglycan, and decorin by fibroblasts, in the liver predominantly by activated stellate cells (myofibroblasts) (32, 33, 36,
37, 46). It has been shown that the level of mRNA for
TGF-
1 and procollagen I, the main extracellular matrix component of
fibrotic liver, is tightly correlated in liver biopsies of patients
with chronic liver disease (11). Besides the fibrogenic effect,
TGF-
1 decreases the degradation of extracellular matrix by
inhibition of metalloproteinases via activation of their inhibitors (tissue inhibitors of metalloproteinases; see Refs. 28, 38, 42). In
vitro data give support for a paracrine and autocrine secretion of
TGF-
1 by activated stellate cells in chronic liver injury (2).
1 for liver fibrosis since increased concentrations of TGF-
1
were found very early in the course of fibrogenesis in these animal
models. In a first transgenic mouse model of hepatic overexpression of
TGF-
1 under control of the albumin promoter, Sanderson et al. (53)
found evidence for a causative role of TGF-
1 for liver fibrogenesis
in vivo, but this model is afflicted with a variety of extraintestinal
complications ascribed to the constant expression of TGF-
1. These
extrahepatic manifestations frequently lead to early death of the
animals due to severe glomerulonephritis, thus limiting the usefulness
of this model. Recently, Clouthier et al. (16) characterized another transgenic mouse model of TGF-
1 overexpression using the regulatory sequences of the phospho-enolpyruvate
carboxykinase (PEPCK) gene. This promoter is postnatally constitutive
and leads to transgene expression in multiple organs, including liver,
kidney, gut, and adipose tissue. Transcription of PEPCK and
PEPCK-driven transgenes can be modulated, both negatively and
positively, by altering the carbohydrate composition of the diet (57).
This animal model has confirmed the results of Sanderson et al. (53)
but has the same limitations.
1, we generated transgenic mice with a
fusion gene consisting of a cDNA coding for the mature form of TGF-
1
under the control of the C-reactive protein (CRP) gene promoter. This
promoter combines the advantage of very low basal expression with the
potential of a massive induction of transgene expression by induction
of an acute phase response, exceeding basal levels >100 times. These
transgenic mice allow the study of the regulation of genes involved in
fibrogenesis and fibrolysis in the adult animal and may serve as a
model for testing of antifibrogenic agents.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 expression
vector and generation of transgenic mice. For transgene
expression, the inducible human CRP gene was chosen. Of several
truncated versions of this gene, construct 79 (41) was kindly provided
by U. Ruether (Hannover, Germany). This version consisted of 1.7 kb of
5' sequences and 3.8 kb of 3' sequences and contained the
BamH
I-Hind III fragment at
1.7 kb
to +3.14 kb as well as the Kpn
I-BamH I fragment from +7.5 kb
to +10.5 kb as described in detail elsewhere (41). This version of the CRP gene is characterized by a low basal level of
expression but a very high degree of inducibility. Out of construct 79 we excised an EcoR I fragment
(position 230 to 2017) containing the coding region of exon 1, the
intron, and a part of exon 2. An oligonucleotide with three single cut
restriction sites (Asc I,
PmeI, and
Sma I) and
EcoR I compatible ends was ligated in the EcoR I site
(5'-TTGGCGCGCCGTTTAAACCCGGG-3'). The 5'
EcoR I site was destroyed by this
insertion. Subsequently, the intron of the human CRP gene was amplified
by PCR. The sequence of the 5'-primer was
5'-TGCTTTTGGCGCGCCAGGTAAG-3' (position 312 to 334) and
included an Asc I restriction site.
The sequence of the 3'-primer was
5'-AGCCTTCCCGGGTATGTCTGTG-3' (position 624 to 603) and
included an Sma I restriction site
(60). The 310-base pair PCR product was ligated in the
Asc
I-Sma I restriction sites of the
construct. Finally, the blunt ended 1.5-kb modified simian TGF-
1
cDNA was inserted in the Sma I site of
the expression cassette (Fig. 1). The
modification of the TGF-
1 cDNA by two cysteine to serine changes at
position 223 and 225 results in biologically active TGF-
1 as had
been described in detail (10, 25). These mutations result in expression
of TGF-
1 that is largely biologically active (51). Transgenic mice
were generated according to standard procedures (26) by microinjection
of a 9.7-kb Sfi I vector-free DNA
fragment (Fig. 1) in fertilized eggs of FVB/NHSD mice. Offspring tail
DNA was isolated 3 wk after birth and was analyzed for the
presence of the transgene by Southern blotting using a 1.3-kb
EcoR I fragment of the human CRP gene
(Fig. 1) or by PCR. The 5'-primer was derived from the human CRP
intron from position 330 to 351 (5'-TAAGGGCCACCCCAGGCTATG-3'; see Ref. 59), and the
3'-primer was derived from the simian TGF-
1 from position 416 to 437 (5'-AGCCGCAGCTTGGACAGGATC-3'; see Ref. 56).
View larger version (8K):
[in a new window]
Fig. 1.
Schematic representation of the 9.7-kb
Sfi I vector-free DNA fragment that
was microinjected in pronuclei of one-cell mouse embryos (FVB/NHSD).
EcoR I fragment (1.3 kb) derived from
the human (h) C-reactive protein gene (CRP) promoter was used for
Southern blot hybridization. , Pseudogen of the hCRP gene; open
area, intron of the hCRP gene; dotted area, 5' cap site and
polyadenylation site of the hCRP gene; hatched area, cDNA for the
active form of simian TGF-
1; cross- hatched area, 5'-upstream
and 3'-downstream sequences of the hCRP gene. Restriction enzymes
in parentheses indicate destruction of the recognition sequence during
vector construction. TGF-
1, transforming growth factor-
1.
Induction of TGF-1 expression in
transgenic mice. Hepatic expression of the
transgene-derived TGF-
1 was achieved by induction of the acute phase
reaction by intraperitoneal injection of 100 µg lipopolysaccharide
(LPS; L-8274; Sigma Chemicals, Heidelberg, Germany). Long-term
induction of transgenic and wild-type animals was achieved by repeated
LPS injections (3 times/wk). For these experiments, 8-wk-old transgenic
and wild-type animals were chosen.
Measurement of TGF-1 plasma levels.
TGF-
1 plasma levels were measured using an ELISA kit (Genzyme)
according to the manufacturer's guidelines. This assay measures both
the mature and the latent forms of TGF-
1. Blood samples were
obtained either by cardiac puncture or for serial testing by incision
of the tail vein. Plasma samples were stored at
20°C until analyzed.
Northern blot analysis. Total cellular
RNA was isolated from various mouse organs by extraction in guanidinium
thiocyanate and centrifugation in cesium chloride as described
previously (13). Aliquots (10 µg) of total RNA were separated on 1%
agarose gels containing 2.2 M formaldehyde and were blotted on nylon
membranes (Hybond N; Amersham, Braunschweig, Germany). Filters were
processed at high stringency as described (14) and were hybridized with the 32P-labeled simian TGF-1
cDNA (1.5 kb). Before electrophoresis, ethidium bromide was added to
the RNA samples to enhance staining to assess the loading and the
transfer efficiency of each RNA sample.
Poly(A)+ mRNA was prepared by using the oligotex mRNA kit (Qiagen, Hilden, Germany). Two micrograms of poly(A)+ mRNA per slot were taken for Northern blot detection of mRNA for procollagen I and procollagen III using a human [32P]cDNA (1.5 kb; see Ref. 34).
Detection of TGF-1 protein expression
in the liver by Western blot hybridization. For Western
blot analysis, liver proteins of transgenic and nontransgenic mice were
separated by SDS-PAGE and were electroblotted on a polyvinylidene
difluoride membrane (Millipore, Eschborn, Germany). For detection, 1 µg/ml of a polyclonal antibody specific for the mature form of
TGF-
1 (AF-101-NA; R&D Systems, Wiesbaden, Germany) or the proregion
of TGF-
1 (anti-LAP, AB-246-NA; R&D Systems) was used. Immune
complexes were visualized using a horseradish peroxidase-conjugated
secondary antibody and chemoluminescence detection (Dianova, Hamburg,
Germany and DAKO, Hamburg, Germany, respectively).
In situ hybridization experiments. In
situ hybridization was performed on frozen liver sections (5 µm).
Cryosections were fixed for 15 min in 4% paraformaldehyde in PBS and
were washed in PBS. Next, slides were preincubated with 20 µg/ml
proteinase K (MERCK, Darmstadt, Germany) in proteinase K buffer for 5 min and were rinsed in PBS, and 0.1 M trietholamine with 0.25% acetic anhydride were added and incubated for 10 min. Thereafter, slides were
washed in PBS, dehydrated in graded ethanol, and air-dried. Sections
were then hybridized at 42°C in hybridization buffer [2× saline sodium citrate (SSC), 5% dextran sulfate, 0.2%
milk powder, and 50% formamide] containing 50 µl
35S-labeled TGF-1 antisense or
sense probes (106 counts per minute). Later (12 h),
sections were rinsed in 2× SSC at 42°C for 30 min and were
treated with 50% formamide and 10 mM dithiothreitol in 2× SSC
for 2 × 15 min. Next, sections were washed for 15 min in 2×
SSC and 10 min in RNase buffer. RNase (20 µg/ml; Behring Diagnostics)
was added and incubated for 30 min. Slides were then washed with RNase
buffer, treated with 50% formamide at 65°C for 15 min, and rinsed
in decreasing concentrations of SSC at 42°C. Thereafter, sections
were dehydrated in graded ethanol, air-dried, and coated with
photographic emulsion (Eastman Kodak, Rochester NY). After exposure,
slides were developed, washed and fixed, and finally counterstained
with hemalum.
Tissues for in situ hybridization were prepared 9 h after induction
with LPS or interleukin (IL)-6, since peak mRNA expression under the
CRP promoter was shown to occur at this time point (see Ref. 15, our
own observation, and Fig.
2A).
|
Bioassay for TGF-1.
The amount of biologically active TGF-
1 in the plasma was quantified
with an assay measuring the inhibition of growth of Mv 1 Lu mink lung
epithelial cells (27). In brief, Mv 1 Lu cells (CCL-64; 30,000 cells/200 µl) were subjected to trypsination, washed in PBS, and
suspended in assay medium [RPMI culture medium (GIBCO, Paisley,
Scotland) supplemented with 1% 1-glutamin, 1%
penicillin, 0.4% mercaptoethanol, and 5% fetal bovine serum
(GIBCO)]. Next, cells were plated at a concentration of 1.5 × 105 cells/ml in 96-well
microtiter plates. After incubation at 37°C for 1 h in a humidified
CO2 incubator, TGF-
1 test
samples and standards of known TGF-
1 concentration (Boehringer
Mannheim) were added to the wells, and cells were cultured for 24 h.
After this preincubation, cells were cultured in the presence of
[3H]thymidine at
37°C for another 24 h. The amount of acid-precipitable radioactivity in the cells exposed to the test samples and TGF-
1 standards was determined with a liquid scintillation counter. All
standards and samples were tested in triplicates. The concentration of
biologically active TGF-
1 was determined by comparison with the
appropriate standard curve.
Histology and immunohistochemistry.
Tissues were fixed in 10% formaldehyde in PBS, embedded in paraffin,
sectioned at 2 µm, and stained with hematoxylin/eosin and Sirius red
by standard methods. Immunhistochemical staining for desmin (Dako
M-0760, Hamburg, Germany) and -actin (Sigma A-2547) was performed
using 2-µm deparaffinized sections. The degree of fibrosis after
repeated inductions of transgene-derived TGF-
1 expression was
evaluated semiquantitatively by the Chevallier score, the scoring
system for liver fibrosis that has been shown to correlate with liver collagen content (12). The scoring index included centrolobular vein,
perisinusoidal collagen, portal tracts, and septa.
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RESULTS |
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Generation of CRP/TGF-1 transgenic
mice. To control the basal level of hepatocellular
TGF-
1 expression, we chose the highly inducible human CRP gene
promoter, a human acute phase protein gene promoter. To bypass the not
fully understood mechanism of activation of latent TGF-
1, we
constructed a fusion gene consisting of a modified cDNA for simian
TGF-
1 and modified portions of the human CRP gene (Fig. 1). In this
variant form of TGF-
1, two cysteines at positions 223 and 225 were
replaced by serines using site-directed mutagenesis leading to
expression of biologically active TGF-
1 without dimerization and
cleavage of the proregion (9). As a consequence, transgene-derived
TGF-
1 is recognized in Western blot analysis by antibodies to the
mature region as well as the proregion (see Fig. 6). Two founder lines
were generated (lines 11 and 12). Screening for transgenic mice was
done by Southern blot hybridization of DNA from tail tissue and by PCR.
Analysis of transgene expression.
Transgene expression was reliably achieved by induction of the acute
phase reaction by application of LPS. The kinetics of the appearance of
simian TGF-1 mRNA in the liver and TGF-
1 protein synthesis
measured as TGF-
1 plasma levels were identical to the published
kinetics of the utilized CRP gene promoter (15). As early as 6 h after
induction with LPS, there was already a marked increase in transgene
mRNA expression with peak values at 9-15 h after induction (Fig.
2A). Thereafter, transgene mRNA
levels declined rapidly and returned to basal concentrations 24 h after
induction. Uninduced transgenic animals showed only a weak basal
transgene overexpression (Fig. 2A).
Northern blot analysis of multiple organs 9 h after induction showed a
strict liver-specific overexpression of simian TGF-
1 mRNA without
any extrahepatic signal (Fig. 2B).
Additionally, we determined the kinetics of TGF-1 plasma levels by
ELISA. The highest TGF-
1 plasma levels were reached 15 h after
induction with LPS with a decline to basal levels after 48 h (Fig.
3). Maximal TGF-
1 plasma concentrations
reached >600 ng/ml in line 12, corresponding to ~100 times basal
plasma concentrations of TGF-
1. Plasma levels of line 11 had the
same kinetics but were considerably lower (50-200 ng/ml at peak
values). Uninduced transgenic animals showed about two- to fivefold
elevated plasma levels of TGF-
1 compared with control animals (20 vs. 5 ng/ml).
|
In a separate set of experiments, IL-6, as a downstream mediator of the
acute phase response, has been tested to induce the expression of
transgene-derived TGF-1 in the liver. However, application of
20-40 mg ip of IL-6 did not lead to a significant induction of
TGF-
1 expression (data not shown).
In situ hybridization for simian TGF-1 mRNA in the liver confirmed
massive hepatocellular accumulation after induction compared with basal
levels (Fig. 4,
B,
B',
C, and
C'). Specific signals were
detected over hepatocytes in a patchy pattern. Induction of age- and
sex-matched wild-type animals with LPS revealed no specific signal
using the simian TGF-
1 antisense probe (Fig. 4,
A and
A').
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Three LPS injections per week as applied for long-term induction of the
TGF-1 transgene expression led to partial tolerance to LPS with
considerably lower TGF-
1 mRNA levels after repeated inductions as
shown in Fig. 5 in a representative
Northern blot (n = 4). TGF-
1 plasma
concentrations 15 h after the last application of LPS were ~50 ng/ml
in these transgenic animals.
|
Western blot analysis of liver homogenates with two different TGF-1
specific antibodies, one recognizing the proregion of TGF-
1 and the
other recognizing the mature region of TGF-
1, was compatible with
ELISA results showing TGF-
1 specific signals in LPS-induced
transgenic animals 15 h after induction. TGF-
1 expression was
undetectable both in uninduced transgenic animals and in induced
wild-type animals (Fig. 6,
lanes 3-6).
|
Bioassay for TGF-1. To
verify the biological activity of the transgene-derived simian
TGF-
1, we tested plasma of uninduced and induced transgenic animals
as well as wild-type animals in a bioassay for TGF-
on the basis of
the growth inhibitory effect of TGF-
1 on mink lung cells. The
concentration of biologically active TGF-
1 was determined by
comparison with the standard curve (Fig.
7). Plasma of induced transgenic animals
led to a significant growth inhibition of mink lung cells compared with
plasma of induced wild-type animals. Uninduced transgenic animals
exhibited only a weak growth inhibition.
|
Procollagen I and III mRNA expression after induction
of the TGF-1 transgene. After induction
of TGF-
1 expression (36 h), corresponding to 21 h after the plasma
peak of TGF-
1, we could demonstrate a marked increase of procollagen
I and procollagen III mRNA expression in the liver (Fig.
8), which was spontaneously downregulated
to baseline expression after 96 h. In contrast, uninduced transgenic
animals and wild-type animals exhibited only a very low basal level of
constitutive expression of procollagen I and procollagen III mRNA.
|
Liver histology. Liver histology 6 wk
after repeated inductions of the acute phase by LPS resulted in a
marked -actin staining of hepatic stellate cells in transgenic but
not wild-type animals, indicative of an activated state of these cells
(Fig. 9).
|
Collagen deposition around individual hepatocytes and a linear pattern
of collagen in the space of Disse could be demonstrated by Sirius red
staining (Fig. 10). The quantitative
evaluation of hepatic fibrosis according to the scoring system of
Chevallier gave a score of 0.1 for the wild type, 2.0 for transgenic
line 11, and 2.6 for transgenic line 12 (P < 0.01). The scores were mainly
due to centrolobular vein sclerosis and perisinusoidal fibrosis.
Consistent with a higher TGF-1 expression of line 12 (Northern blot,
TGF-
ELISA), the fibrosis score was equally higher in line 12. This
signifies a dose-effect relationship between the amount of transgene
expression and the grade of liver collagen content.
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No apparent gross or histological lesions were recorded in transgenic animals 6 wk after repeated inductions of the acute phase (3 times/wk) in kidneys, heart, lung, spleen, muscle, and intestine. As a consequence of repeated LPS injections, fatal glomerulonephritis could be observed in 5-10% of the animals in both wild-type and transgenic animals similarly, as described by others (19, 23, 30). Life expectancy of uninduced transgenic animals was normal.
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DISCUSSION |
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We have established an animal model with inducible overexpression of
mature TGF-1 in hepatocytes. The control of TGF-
1 expression by
the CRP gene promoter combines the advantage of strict liver specificity and low basal expression with a high inducibility (41).
Transiently induced TGF-
1 overexpression led to stellate cell
activation and upregulation of procollagen I and procollagen III mRNA
with subsequent collagen deposition in the liver, giving strong in vivo
evidence for a stimulatory role of TGF-
1 in liver fibrogenesis.
Although a significant amount of hepatic TGF-
1 is derived from
stellate cells and Kupffer cells, there is now convincing evidence
that, at least under pathological situations, hepatocytes are able to
produce or secrete TGF-
1 (4, 6, 17, 29, 33). This was a further
rationale for choosing the hepatocellular CRP gene promoter for
TGF-
1 expression in our model.
Even though serum amyloid proteins rather than CRP are the major acute
phase proteins in mice (43, 61), the expression of the human CRP gene
is regulated in transgenic mice as it is regulated in humans (15).
Corresponding to this finding, we could show by Northern blot
hybridization and in situ hybridization a massive increase of
transgene-derived TGF-1 mRNA in the livers of transgenic animals
after induction of an acute phase response by LPS. By Western blot
analysis of liver homogenates with TGF-
1 antibodies or by ELISA for
TGF-
1 in plasma, we could demonstrate a massive overexpression of
TGF-
1 protein both in the liver and the plasma. Increased plasma
levels result from TGF-
1 synthesized in the liver and released in
the circulation, reaching >100 times basal plasma levels. The local
concentrations of TGF-
1 in the liver are therefore probably much
higher than the measured plasma levels. The kinetics of
transgene-derived TGF-
1 mRNA induction and the kinetics of TGF-
1
plasma levels were identical to the CRP expression kinetics published
by Ciliberto et al. (15).
After the peak of TGF-1 expression, we could demonstrate a marked
transient induction of procollagen I and procollagen III mRNA in the
liver of transgenic but not of wild-type animals. To our knowledge,
this demonstrates for the first time a direct causal relationship
between TGF-
1 overexpression in the liver and consecutive
procollagen I and procollagen III expression in vivo. With this
inducible animal model, it will be possible to characterize factors or
pharmaceuticals leading to downregulation of procollagen expression in
the liver.
To achieve repetitive increased TGF-1 expression in the liver, we
treated transgenic and wild-type animals three times per week with LPS.
Long-term induction of transgenic animals with LPS over 6 wk led to an
attenuated transgene response, marking the development of partial
tolerance to repeated LPS injections as reported previously (18, 20,
52, 58). Because TGF-
1 has been shown to be a possible mediator of
LPS tolerance in vitro, the phenomenon of LPS tolerance might be
aggravated by transgenic TGF-
1 in our animals (45). However, mRNA
for transgene-derived TGF-
1 in the liver remained clearly
detectable. Future studies have to show if LPS dose escalation can
maintain comparable levels of induction of transgene-derived TGF-
1,
as had been proposed in a comparable transgenic study (50).
Six weeks after repeated treatments with LPS, wild-type animals showed
no significant changes in liver morphology. Liver histology of
uninduced, age-matched transgenic animals was equally normal. However,
induced transgenic animals showed a marked activation of hepatic
stellate cells. As the most important biological effect of TGF-1
overexpression, transgenic mice showed beginning hepatic fibrosis after
6 wk of repeated inductions of TGF-
1 expression. The fibrotic
process was characterized by increased perisinusoidal deposition of
collagen. It is conceivable that LPS induced release of tumor necrosis
factor (TNF), or other cytokines potentiate the profibrogenic effect of
TGF-
1 in the liver of transgenic animals, even though LPS alone had
no profibrinogenic effects in control mice. In this context, it has
been shown that TGF-
1 and TNF have some synergistic effects on
stellate cell activation in vitro (3). Furthermore, Greenwel and
Rojkind (22) found an accelerated development of liver fibrosis in
carbon tetrachloride-treated rats by the weekly induction of the acute
phase response.
To avoid potential side effects and tolerance to LPS, we tested IL-6 as
one of the downstream mediators of the acute phase response. However,
IL-6 alone did not lead to a significant induction of transgene-derived
TGF-1, although the acute phase response was induced. This confirms
recently published data showing that IL-6 is necessary, but not
sufficient, to induce the human CRP gene in transgenic mice (59).
Interestingly, we did not observe significant extrahepatic pathology in
kidneys, spleen, heart, lung, muscle, and intestine in long-term
induced transgenic animals. This is in marked contrast to the
albumin/TGF-1 transgenic model of Sanderson and colleagues (53).
Transgenic mice with constitutive, hepatocellular overexpression of
activated TGF-
1 under control of the albumin promoter also exhibited
limited hepatic fibrosis but suffered from marked extrahepatic organ
manifestations even though TGF-
1 plasma levels were relatively low
compared with our model (peak values 40 ng/ml vs. >600 ng/ml). What
are the reasons for this difference? First of all, the albumin promoter
is a constitutive promoter leading to a constant TGF-
1 overexpression during embryogenesis and thus probably during a particularly vulnerable phase of organ differentiation. In addition, TGF-
1 plasma levels in the albumin/TGF-
1 transgenic animals were
highest during the first 4 wk postpartum and might be even higher in
utero. Furthermore, the albumin promoter is not liver specific during
embryogenesis. Albumin mRNA and protein have been found prenatally in
intestine, kidney, lung, and liver (44, 55). Postnatally, albumin
promoter activity was demonstrated in kidneys until 3 wk postpartum,
which could explain the high percentage of fatal glomerulonephritis in
this model (53). The recently published PEPCK/TGF-
1 transgenic mouse
model (16) has the same limitations as the albumin/TGF-
1 transgenic
model. The promoter is postnatally constitutive and not liver specific, with high levels of expression in kidney, gut, adipose tissue, and a
variety of other organs (57). In contrast, our inducible model gives us
the opportunity to study effects of hepatic TGF-
1 overexpression in
healthy adult animals. This is of pathophysiological significance,
since liver fibrosis is mainly an acquired disease of adulthood.
The development of only mild fibrosis that we observed in repeatedly
induced transgenic animals might have several reasons. LPS tolerance
with significantly lower expression of transgene-derived TGF-1 might
be one important reason. Additionally, factors that abolish the effect
of TGF-
1 could be upregulated. We have some preliminary evidence
that antifibrogenetic proteins are upregulated in LPS-stimulated
transgenic animals compared with LPS-stimulated wild-type animals.
Other antifibrotic mechanisms might also counteract the effects of
TGF-
1. Finally, the limited fibrosis observed suggests that TGF-
1
is only one important piece in the puzzle of liver fibrogenesis.
Additional factors such as necroinflammation and probably longer
periods of continuous TGF-
1 exposure may be necessary for the
development of full-blown fibrosis or cirrhosis. In consequence, future
experiments should examine the effects of hepatotoxic chemicals like
carbon tetrachloride and/or ethanol in these transgenic animals to
study fibrogenesis in the setting of necroinflammation. Due to the high
degree of inducibility, this animal model will also allow the study of
the reversibility of liver fibrosis. Finally, it may serve as a model
for the testing of antifibrinogenic agents.
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ACKNOWLEDGEMENTS |
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We thank C. Waldmann and M. Fischer for excellent technical assistance. We thank U. Ruether (Hannover, Germany) for supplying a cDNA clone of the human CRP promoter.
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FOOTNOTES |
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This work was funded by Deutsche Forschungsgemeinschaft (SFB 311 and Lo 368/4-1) and the Boehringer Ingelheim Foundation.
Parts of this paper were presented at the 48th Annual Meeting of the American Association for the Study of Liver Disease in Chicago, IL, November 1997.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. W. Lohse, I. Dept. of Medicine, Johannes Gutenberg Univ., Langenbeckstrasse 1, 55101 Mainz, Germany. (E-mail: lohse{at}mail.unimainz.de).
Received 2 September 1998; accepted in final form 3 December 1998.
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