International Immunology, Vol. 11, No. 9, pp.

1491–1500 © 1999 The Japanese Society for Immunology

Immunopathogenesis of hepatic fibrosis in

chronic liver injury induced by repeatedly
administered concanavalin A
Kiminori Kimura, Kazuki Ando, Hiroo Ohnishi, Tetsuya Ishikawa2,
Shinichi Kakumu2, Masao Takemura1, Yasutoshi Muto and Hisataka Moriwaki
First Department of Internal Medicine and 1Department of Laboratory Medicine,Gifu University School of
Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan
2First Department of Internal Medicine, Aichi Medical University, Nagakute-cho, Aichi district,
Aichi 480-1100, Japan

Keywords: concanavalin A, hepatic fibrosis, hepatitis, immunology, in vivo animal model, transforming
growth factor-β

Abstract
Liver fibrosis is commonly observed in chronic liver disease. However, the immunological
mechanisms underlying hepatic fibrosis due to chronic inflammation are not well defined, mainly
because suitable experimental models have not been established. We have found that weekly i.v.
administration of concanavalin A (Con A) in BALB/c mice brought about a striking alanine
aminotransferase increase, resulting in piecemeal necrosis with bridging fibrosis in the
parenchyma. Using this fibrosis model, we demonstrated the kinetics of cytokine mRNA
expression in liver. Transforming growth factor (TGF)-β1, TGF-α, basic fibroblast growth factor
(bFGF) and hepatocyte growth factor mRNAs were up-regulated after each Con A administration.
Furthermore, either anti-IFN-γ, anti-tumor necrosis factor (TNF)-α or anti-TGF-β mAb given together
with Con A markedly inhibited the development of hepatic fibrosis. Treatment with either anti-IFN-γ
or anti-TNF-α mAb also completely prevented hepatic injury; in contrast, treatment with anti-TGF-β
mAb did not. The treatment with anti-TGF-β mAb did not affect the levels of hepatic mRNAs for
either IFN-γ or TNF-α after Con A injection. Treatment with either anti-IFN-γ or anti-TNF-α did not
affect the expression levels of TGF-β in the liver. In conclusion, the continuous presence of both
severe liver damage and up-regulation of TGF-β synthesis is necessary to induce hepatic fibrosis
in this model.

Introduction
Hepatic fibrosis is a common feature of chronic hepatitis and
chronic liver injury leads to liver cirrhosis. Regardless of
the causes, the fibrosis is characterized by an increase in
extracellular matrix constituents that collectively form hepatic
scars (1). Liver cirrhosis, represented for hepatic fibrosis, is
clinically very important as high risk conditions underlie the
development of hepatocellular carcinomas (HCC), but the
immunological mechanisms for hepatic fibrosis are not well
defined. For prevention of HCC, an understanding of such
mechanisms is essential. In human cases, it has been shown
that hepatic fibrosis is caused after the death of hepatocytes
by immunological mechanisms with infection with hepatitis
viruses.
Although there have been reports of experimental fibrosis
models established by repeated administration of carbon
tetrachloride (2), ligation of the bile duct (3) and porcine
serum (4), these experimental models are generally unsuitable
for analyzing the mechanisms of human hepatic fibrosis, since
these models do not involve immunological responses, such
as lymphocyte infiltration and IFN-γ synthesis, compatible for
human cases.
Recently it has been shown that hepatitis B surface antigen
(HBsAg)-specific cytotoxic T lymphocytes (CTL) cause a fatal
necroinflammatory liver disease in HBsAg transgenic mice
(5,6). This model revealed that antigen-non-specific ampli-
fication mechanisms play a major role in spreading the liver
necrosis (6,7). It has been reported that hepatitis B virus
(HBV)-specific CTL are actually induced in patients with acute

Correspondence to: K. Ando

Transmitting editor: K. Okumura Received 21 April 1999, accepted 27 May 1999
1492 Mechanisms of Con A-induced hepatic fibrosis
hepatitis B (8–11) and thus the HBsAg transgenic mouse
model may have essential similarities to human hepatitis.
However, since adoptively transferred HBsAg-specific CTL
abolish the HBV gene and antigen expression in the liver
(12,13), a 4 week interval of CTL administration is required to
induce an equal level of liver injury in this model. Thus, it has
been difficult to establish liver fibrosis using this mouse model,
although it is speculated that continuous hepatocellular injury
may induce liver fibrosis. With another approach, it has been
reported that liver-specific expression of IFN-γ in transgenic
mice causes chronic active hepatitis, in which initial necroin-
flammatory changes are followed by lymphoid cell infiltration
in the portal areas (14). However, since the mouse liver has
a remarkable regenerative capacity for individual hepato-
cytes, a mouse model of hepatic fibrosis following immunolo-
gically induced hepatocellular injury has not yet been
produced. According to these results, it has been thought to
be difficult to establish a liver fibrosis model after hepatocellu-
lar injury mediated by immunological mechanisms.
We now report that concanavalin A (Con A)-induced hepat-
itis develops hepatic fibrosis after repeated liver injury and
the mechanisms are mainly based on the up-regulation of
transforming growth factor (TGF)-β1 expression in the liver.
Methods
Mice
Female BALB/c mice (7–10 weeks old; weight 25–30 g) were
obtained from Japan SLC (Shizuoka, Japan).
Disease model
Con A (Sigma, St Louis, MO) was dissolved in pyrogen-free
PBS (Sigma) at a concentration of 1 mg/ml and i.v. injected
into BALB/c mice at a dose of 0.3 mg/mouse once a week.
Serum samples from individual mice were obtained from the
funds oculi vein at indicated time points after each Con A
injection. Hepatocellular injury was monitored biochemically
by measuring serum alanine aminotransferase (sALT) activity.
For the histopathological evaluation, mice were sacrificed 1
week after the final injection of Con A. Liver tissues were fixed
in 10% neutral-buffered formalin and embedded in paraffin.
Sections of 5 µm thickness were stained with hematoxylin &
eosin, Azan or silver for light-microscopic evaluation.
Serum levels of IFN-γ and tumor necrosis factor (TNF)-α
The serum levels of IFN-γ and TNF-α were monitored using
commercially available ELISA kits (Gibco/BRL, Gaithersburg,
MD and Genzyme, Cambridge, MA respectively). The detect-
able lower limits of sensitivity of the kits were 125 and
35 pg/ml respectively. To evaluate the role of IFN-γ and TNF-
α in the fibrosis process, Con A was administered at 24 h
after the i.p. injection of hamster mAb H22 and TN3 19.12
specific for murine IFN-γ and TNF-α respectively, which were
generously provided by Dr Robert D. Schreiber (Department
of Pathology, Washington University School of Medicine, St
Louis, MO). Purified hamster IgG (Organon, West Chester,
PA) was used as a control antibody.
RNA preparation
Frozen liver tissue was mechanically pulverized and total
hepatic RNA was isolated for the analysis of cytokine mRNA
expression at the indicated time points after Con A injection.
Total RNAs were isolated by the guanidinium isothiocyanate–
phenol–chloroform method using RNAzol (Tel-Test,
Friendswood, TX). In brief, tissue samples were homogenized
with RNAzol and 0.2 ml chloroform was added to 2 ml of
homogenate. The samples were shaken vigorously for 15 s
and incubated for 5 min on ice, and then centrifuged at
12,000 g for 15 min. The aqueous phase was transferred to
a fresh tube, mixed with an equal volume of isopropanol and
placed for 15 min on ice. After centrifugation at 12,000 g for
15 min, the RNA pellet was washed once with ice-cold 75%
ethanol with vortexing, centrifuged at 7500 g for 5 min, dried
for 10 min and dissolved in diethylpyrocarbonate (Sigma)-
treated RNase-free solution. Total RNA contents of the
samples were estimated spectrophotometrically by
absorbance at 260 nm. Purity of RNA was determined from
the 260/280 absorbance ratio.
RT-PCR analysis
Total RNA (1 µg) was reverse transcribed into cDNA by using
AMV reverse transcriptase (Boehringer, Mannheim, Germany)
and the cDNAs were amplified by PCR. Briefly, 1 µg of total
RNA was denatured by heating for 10 min at 70°C and
added to a mixture containing 2 µl of oligo-dT (0.5 mg/ml)
(Boehringer), 6 µl of 5-fold concentrated reverse transcriptase
buffer, 2 µl of DTT (Boehringer; 0.1 mol/l), 1 µl of dNTP
(Boehringer; 10 mmol/l) and 1 µl of reverse transcriptase.
Each cDNA synthesis was performed for 1 h at 42°C and
stopped by incubation for 10 min at 98°C. PCR was performed
in a volume of 50 µl containing 400 pmol/l of each primer, 2
µg of cDNA, Tris–HCl (10 mmol/l, pH 8.3), KCl (50 mmol/l),
MgCl2 (1.5 mmol/l), dNTP (0.2 mmol/l) and 2 U of Taq DNA
polymerase (Boehringer). Thermal cycle conditions were 94°C
for 45 s for denaturing, 60°C for 45 s for annealing and 72°C
for 2 min for extension. The number of amplification cycles
was 28 for each set of primers. For each set of primers,
dilutions of cDNA were amplified for 20, 23, 25, 28, 30, 32,
34, 36 and 38 cycles to define optimal conditions for linearity
and to permit semiquantitative analysis of signal strength. After
the last cycle of amplification, the samples were incubated for
7 min at 72°C. The PCR products were electrophoresed on
1.2% agarose gels and stained with ethidium bromide. To
ensure that contaminating DNA had not been amplified, PCR
without reverse transcriptase was simultaneously demon-
strated. Sense and anti-sense primers were based on Clontech
(Palo Alto, CA) Amplimer sets for mouse IFN-γ, TNF-α,
TGF-β1 and G3PDH. In addition, TGF-α, acidic fibroblast
growth factor (aFGF), basic FGF (bFGF) and hepatocyte
growth factor (HGF) were investigated. The sense and anti-
sense primers were 59-TGC CCA GAT TCC CAC ACT-39 and
59-TGG ATC AGC ACA CAG GTG for TGF-α, 59-CGG GGG
CCA CTT CTT GAG GA-39 and 59-ACC GGG AGG GGC AGA
AAC AA-39 for aFGF, 59-TTC CCA CCA GGC CAC TTC A-39
and 59-TGC CCA GTT CGT TTC AGT GC-39 for bFGF, and
59-CCG AGG CCA TGG TGC TAC A-39 and 59- CTC GGA
TGT TTG GGT CAG TTG-39 for HGF respectively. Quantitation
of IFN-γ, TNF-α and TGF-β1 mRNAs was performed by

competitive PCR using the PCR Mimic Protocol (Clontech).
IFN-γ, TNF-α and TGF-β1 competitor primers yielding product
sizes of 500, 500 and 390 bp respectively were used in
each reaction. Furthermore, G3PDH competitor primers was
constructed using a PCR Mimic construction kit (Clontech).
Role of TGF-β1 in hepatic fibrosis
To evaluate the role of TGF-β in the development of liver
fibrosis, the monoclonal neutralizing antibody to TGF-β1, 2
and 3, purchased from Genzyme (Cambridge, MA), was
simultaneously administered (200 µg/mouse/each injection)
i.p. at the time of each Con A injection. Sera and liver tissues
were collected at several time points after the administration
of Con A and anti-TGF-β mAb for analysis of both hepatic
mRNA expressions and serum levels of IFN-γ and TNF-α.
Histopathological examination was also performed using the
liver tissues.
Statistical analysis
Values were expressed as mean 6 SD. Differences between
experimental and control groups were analyzed by the Krus-
kal–Wallis test followed by Scheff’s F-test.
Results
The evaluation of hepatocellular injury induced by repetitive
Con A injections
After Con A was i.v. injected into BALB/c mice at a dose of
0.3 mg/mouse, sALT activities were measured at indicated
time points (Fig. 1A). sALT levels began to rise at 4 h (434 6
138 IU/l) and reached a peak at 24 h (4840 6 1194 IU/l). The
levels then rapidly decreased within 48 h (490 6 130 IU/l).
With weekly administrations of Con A, as shown in Fig. 1(B),
Fig. 1. Hepatocellular injury during early phase after Con A injection
(A). BALB/c mice were i.v. injected Con A at a dose of 0.3 mg/mouse.
Hepatocellular injury was monitored by analyzing sALT levels 0, 4, 8,
16, 24, 36 and 48 h after Con A injection. Each data and error bar
represent the mean of results in five mice and the SD respectively.
Effect of repeated administration of Con A on hepatocellular injury
(B). BALB/c mice were i.v. injected with Con A at a dose of 0.3 mg/
mouse. sALT levels were analyzed 24 h after each weekly Con A
injection. Each bar and error bar represent the mean of results in
seven mice and the SEM respectively.
Mechanisms of Con A-induced hepatic fibrosis 1493
striking increases of sALT were observed 24 h after each
injection.
Histopathological analysis of hepatic fibrosis
To assess the evolution of pathological changes in livers due
to weekly Con A injections, liver tissues were derived from
mice before Con A injection, 24 h after the first injection,
1 week after the second injection and 1 week after the
fourth injection. Twenty-four hours after first Con A injection,
hepatocellular necrosis had become widespread throughout
the lobule (Fig. 2B). No evidence of hepatic fibrosis was
observed at this period. No hepatocellular necrosis and
hepatic fibrosis were found 1 week after the second Con A
injection (Fig. 2C). However, as shown in Fig. 2(D), bridging
fibrosis in the parenchyma with hepatocellular necrosis was
detectable 1 week after the fourth Con A injection by Azan
and silver staining (inset).
Role of IFN-γ and TNF-α
Since it has been reported that both IFN-γ and TNF-α play
critical roles in Con A-induced liver injury (15,16), we investi-
gated changes in both serum levels and hepatic mRNA
expression of IFN-γ and TNF-α at several time points after
Con A injection. Serum levels of IFN-γ gradually increased
and reached a peak 12 h after a single Con A injection (4328
6 1109 pg/ml). Serum levels of TNF-α strikingly increased
and reached a peak within 2 h (1122 6 105 pg/ml) (data not
shown). Quantitative RT-PCR performed using liver tissues
also revealed up-regulation of hepatic mRNA expression of
both IFN-γ and TNF-α, reaching peaks within 4 h after Con A
injection (data not shown).
In order to reveal the role of IFN-γ and TNF-α in hepatic
fibrosis, we investigated the protective effects of passive
immunization with anti-IFN-γ mAb and anti-TNF-α mAb in this
model. Anti-IFN-γ mAb or anti-TNF-α mAb (150 µg/mouse)
were administrated i.p. 24 h before each Con A injection.
As shown Fig. 3(A), the serum ALT levels were markedly
suppressed by the administration of either anti-IFN-γ mAb or
anti-TNF-α mAb after every Con A injection. Histopathological
examination similarly revealed inhibition of hepatocellular
injury and hepatic fibrosis (Fig. 3B and C) at 1 week after the
fourth Con A injection. To evaluate whether the inhibitory
effects were due to independent pathways, the serum levels
of IFN-γ or TNF-α were monitored after Con A injection with
anti-TNF-α mAb or anti-IFN-γ mAb. As shown in Fig. 4(A and
B), the levels of IFN-γ showed a 84% reduction in the peak
after Con A injection with anti-TNF-α mAb, as compared with
the Con A injection alone. Similarly, an 87% reduction in the
peak of TNF-α was observed after Con A injection with anti-
IFN-γ mAb, as compared with the Con A injection alone.
Cytokine gene expression in liver after Con A administration
To determine whether cytokines contribute to the hepatic
fibrosis, we analyzed the mRNA expression levels of TGF-α,
bFGF, HGF, aFGF and TGF-β1, reported to affect tissue
fibrogenesis (17–22) by either semiquantitative or competitive
RT-PCR after Con A administration. As shown in Fig. 5(A),
mRNA expression of TGF-α, bFGF and HGF was up-regulated
after Con A injection (peak 5 4, 24 and 24 h respectively).
In contrast, aFGF was negatively regulated. Competitive
1494 Mechanisms of Con A-induced hepatic fibrosis

Fig. 2. Histopathological evaluation of the livers of Con A-treated mice. BALB/c mice were i.v. injected with Con A at a dose of 0.3 mg/mouse
once per week. Histopathological characteristics of liver without Con A injection (A), 24 h after the first injection (B), 1 week after the second
injection (C) and 1 week after the fourth injection (D). Hematoxylin & eosin staining was performed for (A) and (B), Azan staining for (C), and
Azan staining and silver staining (inset) for (D). Note the presence of hepatic fibrosis (arrowheads) in (D). Original magnification 3200.
Representative figures derived from analyses of four mice are shown in each time point.

RT-PCR revealed that mRNA expression of TGF-β1 was up-
regulated and reached a peak 48 h after Con A injection (Fig.
5B). These results indicated that most of the cytokine’s mRNAs
except for aFGF were up-regulated after Con A injection.
Prevention of Con A-induced hepatic fibrosis by anti-TGF-
β mAb
Since it has been recently reported that the hepatic expression
of TGF-β1 mRNA is markedly up-regulated in patients with
liver cirrhosis, as compared with both normal control and
chronic hepatitis (23), we focused on the role of TGF-β1
among the cytokines studied above in the development of
hepatic fibrosis in this mouse model.
As a first step to determine whether TGF-β1 affects the liver
injury induced by Con A, we monitored sALT activities 24 h
after each Con A injection either with or without administration
of anti-TGF-β mAb from weeks 1 to 4. There was no significant
difference in sALT levels between the two groups at any time
points (Fig. 6A). In contrast, histopathological examination
revealed that the administration of anti-TGF-β1 mAb markedly
inhibited hepatic fibrosis (Fig. 6C).
Relationship between TGF-β and IFN-γ or TNF-α in Con A-
induced hepatic fibrosis
To determine the relationships between the cytokines pre-
venting hepatic fibrosis in this model, serum levels and
hepatic mRNA expression of IFN-γ and TNF-α were analyzed
after Con A injection, with or without simultaneous anti-TGF-
β mAb treatment. As shown in Fig. 7(A), serum levels of
IFN-γ, but not TNF-α, were significantly increased by the
simultaneous treatment of anti-TGF-β mAb 4 h after Con A
injection. Reflecting these results, hepatic mRNA expression
of IFN-γ was enhanced; in contrast, that of TNF-α was not
enhanced by the simultaneous treatment of anti-TGF-β mAb
(Fig. 7B). Treatment with either anti-IFN-γ or anti-TNF-α mAb
did not affect the expression levels of TGF-β1 mRNA in liver
after Con A injection (Fig. 7B).
Discussion
Fibrosis is an important component of advanced chronic
inflammatory liver disease. Although the changes in hepatic
 Mechanisms of Con A-induced hepatic fibrosis 1495

Fig. 3. Role of IFN-γ and TNF-α in hepatocellular injury and hepatic fibrosis. Sera and liver tissues were sampled from BALB/c mice 1 week
after each weekly Con A injection (0.3 mg/mouse). Control rat-IgG, anti-IFN-γ or anti-TNF-α mAb (150 µg/mouse) were administered i.p. 24 h
before weekly Con A injections. Hepatocellular injury was monitored by analyzing sALT levels. Pretreatment with either anti-IFN-γ or anti-TNF-
α mAb completely inhibited hepatocellular injury as compared with that with control rat-IgG. Each value represents the mean 6 SD of results
in three mice. The point marked (**P , 0.001) is significantly lower than a group of mice treated with control antibody (A). Histopathological
examinations revealed that administration of either anti-IFN-γ (B) or anti-TNF-α mAb (C) (150 µg/mouse) completely inhibited the development
of hepatic fibrosis 1 week after the fourth weekly Con A injection. Azan staining was performed for (B) and (C). Original magnification 3100.
Representative figures derived from the analysis of three mice are shown in each group.

structures that occur in liver cirrhosis have been described
in detail (24), the immunopathogenesis of fibrosis in this
process is largely unknown. In order to elucidate the under-
lying mechanisms, an appropriate animal model with essential
similarities to patients with chronic hepatitis should be estab-
lished. Ideally, the animal model should have the following
features: (i) it should be based upon immunological mechan-
isms, (ii) hepatic fibrosis should be induced following continu-
ous liver injury and (iii) the inducer of hepatic fibrosis is should
not be a direct hepatotoxin. However, no animal model that
satisfies these criteria has been established.
As cited above, the animal model of liver injury induced by
HBV-specific CTL in HBV transgenic mice has similarities to
human cases (6). However, liver injury cannot be continuously
induced, since the expression of HBsAg is down-regulated
by the CTL.
It has been recently described that Con A-induced experi-
mental liver injury in mice is mediated by activated T cells
(25,26). Con A is a lectin from jack bean and is known as a
T lymphocyte mitogen in vitro. Tiegs et al. reported that acute
liver injury was induced within 8 h after i.v. injection of Con A
in BALB/c mice. The organ specificity was attributed to the
preferential binding of Con A in the liver. Because immunode-
ficient mice (SCID or athymic mice) or mice treated with
immunosuppressive drugs such as cyclosporine, FK506 and
corticosteroids do not develop hepatitis after Con A injection,
it is accepted that the model requires T cell activation. Cell
depletion experiments identified CD41 T cells as the effector
cells (25). Furthermore, neutralization experiments revealed
that TNF-α and IFN-γ play critical roles in the development of
Con A-induced hepatitis (15,16). Since these results indicate
that Con A-induced hepatitis is based upon immunological
mechanisms and has similarities to chronic inflammatory liver
disease in man, Con A-induced hepatitis model was thought
to be one of the potent models to induce liver fibrosis mediated
by hepatocellular injury.
In the present study, we established a hepatic fibrosis model
with repeated administrations of Con A (Fig. 2). Hepatocellular
injury was consistently observed in terms of sALT elevation
24 h after each Con A injection (Fig. 1A and B) following an
1496 Mechanisms of Con A-induced hepatic fibrosis

Fig. 4. The effect of anti-TNF-α mAb and anti-IFN-γ mAb on the kinetics of serum levels of IFN-γ (A) and TNF-α (B) after Con A injection.
Control rat IgG, anti-IFN-γ or anti-TNF-α mAb (150 µg/mouse) were administered i.p. 24 h before a single Con A injection (0.3 mg/mouse).
Hepatocellular injury was monitored by analyzing sALT levels. Each value represents the mean 6 SD of results in five mice. The point marked
(**P , 0.001) is significantly higher than a group of mice treated with either anti-TNFα mAb or anti-IFNγ mAb.

Fig. 5. Cytokine expression in the liver after Con A administration. Hepatic TGF-α, bFGF, HGF and aFGF mRNA levels were analyzed by
semiquantitive RT-PCR at indicated time points after Con A injection (0.3 mg/mouse) (A). TGF-β1 mRNA was assessed by competitive RT-PCR
(B). Liver samples were obtained at the times shown. The number of amplification cycles was 28 for each set of primers. Dilutions of cDNA
were amplified for 20, 23, 25, 28, 30, 32, 34, 36 and 38 cycles to define optimal conditions for linearity and to permit semiquantitative analysis
of signal strength. Representative figures from the analyses of four mice are shown in each group. G3PDH was used as the internal control
(A). The quantitation of TGF-β1 mRNA was performed by competitive RT-PCR using the PCR Mimic protocol (Clontech). The concentrations
of competitors were as follows: a 5 5310–1, b 5 1.5310–1, c 5 6.25310–2 and d 5 5310–2 amol/µl.

increase in serum levels of both IFN-γ and TNF-α (Fig. Biosynthesis and turnover of the extracellular matrix are
2A). Since hepatocellular injury was completely inhibited by thought to be regulated by different cytokines and growth
pretreatment with either anti-IFN-γ or anti-TNF-α mAb (Fig. factors, including TGF-β, FGF, insulin-like growth factor, TGF-
3A), cytokine involvement could be proven in this model. α and HGF. In particular, TGF-β has been identified as the
 Mechanisms of Con A-induced hepatic fibrosis 1497

Fig. 6. Prevention of Con A-induced hepatic fibrosis by anti-TGF-β mAb. Either bovine IgG (200 µg/mouse) or anti-TGF-β mAb (200 µg/mouse)
was administered i.p. into BALB/c mice simultaneously with each Con A injection (0.3 mg/mouse). Hepatocellular injury was monitored by
analyzing sALT levels 24 h after each weekly Con A injection. Each value represents the mean 6 SD of results in three mice (A). Histopathological
evaluation of the liver 1 week after the fourth Con A injection with either control bovine IgG (200 µg/mouse, B) or anti-TGF-β mAb (200 µg/
mouse, C). Azan staining was performed for (B) and (C). Note the marked inhibition of hepatic fibrosis in (C) as compared with that in (B).
Original magnification3200. Representative figures derived from the analyses of three mice are shown in each group.

most potent fibrogenic mediator, contributing to immune and
inflammatory responses, tissue repair, and cell growth and
differentiation (27). Thus, it is speculated that TGF-β is one
of the major causes of fibrosis in hepatic cirrhosis, glomerulo-
nephritis and idiopathic pulmonary fibrosis (1,28,29).
In the present study, expression of TGF-α, HGF, bFGF
and TGF-β1 mRNAs was up-regulated after each Con A
administration (Fig. 5A and B).
Using an experimental model of schistosomiasis in mice,
Czaja et al. similarly found an increase in the content of
hepatic TGF-β1 mRNA in infected animals, in parallel with an
increase in type I procollagen mRNA (30). Nakatsukasa et al.
also demonstrated a positive correlation between TGF-β1
mRNA, and procollagen I, III and IV mRNAs in the livers of
rats with carbon tetrachloride-induced hepatic fibrosis (31).
It has furthermore been reported that hepatic TGF-β1 mRNA
was enhanced in patients with chronic liver disease (23).
In two recent studies, TGF-β1 activity has been successfully
suppressed in vivo with anti-TGF-β1 antibodies, resulting in a
significant decrease in excess extracellular matrix deposition
(32,33). In the present study, simultaneous administration of
neutralizing TGF-β mAb with each Con A injection markedly
reduced hepatic fibrosis and extracellular matrix formation
(Fig. 6B), although it exerted no significant effects on hepato-
cellular injury. On the other hand, treatment with either anti-
IFN-γ or anti-TNF-α mAb completely prevent hepatic injury
(Fig. 3A). Thus, while treatment with anti-IFN-γ, anti-TNF-α or
anti-TGF-β mAb markedly prevented hepatic fibrosis, the
prevention was based on different mechanisms. Furthermore,
serum levels of IFN-γ were significantly increased by the
simultaneous treatment of anti-TGF-β mAb 4 h after Con A
injection (Fig. 7A), but no equivalent influence on TNF-α was
observed. Reflecting these results, hepatic mRNA expression
of IFN-γ was enhanced; in contrast, that of TNF-α was not
enhanced by the simultaneous treatment of anti-TGF-β mAb
(Fig. 7B). Thus, the mechanism of prevention of hepatic
fibrosis by anti-TGF-β mAb is clearly not through the down-
regulation of either IFN-γ or TNF-α synthesis. Since, treatment
with either anti-IFN-γ or anti-TNF-α mAb did not affect hepatic
mRNA expression of TGF-β1 after Con A injection (Fig. 7B),
as compared with Con A injection alone, their influences are
separate. This result indicated that TGF-β synthesis is not
1498 Mechanisms of Con A-induced hepatic fibrosis

Fig. 7. Relationships between TGF-β, IFN-γ and TNF-α in Con A-induced hepatic fibrosis. Serum levels of IFN-γ and TNF-α were analyzed
after Con A injection either with or without simultaneous treatment of anti-TGF-β mAb (i.p., 200 µg/mouse) at indicated time points. Serum
levels of IFN-γ were significantly increased by the simultaneous treatment of anti-TGF-β mAb 4 h after Con A injection (**P , 0.01). In contrast,
serum levels of TNF-α were significantly decreased 8 h after Con A injection (*P , 0.05). Each value represents the mean 6 SD of results in
three mice (A). The effects of anti-TGF-β mAb on the expression of IFN-γ and TNF-α mRNA in liver after Con A injection (B, upper panel).
Liver tissues were derived from BALB/c mice 4 h after Con A injection (at a time of peak expression of either IFN-γ and TNF-α mRNA; data
not shown). The effect of either anti-IFN-γ or TNF-α mAb on the expression of TGF-β mRNA in liver after Con A injection (B, lower panel). Liver
tissues were derived from BALB/c mice 48 h after Con A injection (at a time of peak expression of TGF-β mRNA; see Fig. 5B). The number of
amplification cycles was 28 using various cytokine-specific primers and quantitation of mRNAs was performed by using the PCR
Mimic protocol (Clontech). The concentrations of competitors were as follows: a 5 5310–1, b 5 1.5310–1, c 5 6.25310–2, d 5 5310–2,
e 5 1.5310–2, f 5 5310, g 5 1.5310, and h 5 6.25310–1, attomol/µl (B). Results are from the analyses of three mice in each group.

sufficient to develop hepatic fibrosis. In addition, since treat-
ment with anti-TGF-β mAb prevented fibrosis, repeated liver
injury is also not enough to induce hepatic fibrosis.
An interesting report has recently been published regarding
the correlation between TGF-β1 and liver fibrosis, (34). The
authors established TGF-β1 transgenic mice and indicated
that marked collagen deposition was shown localized to the
sinusoids and around periportal areas. However, liver fibrosis
such as bridging fibrosis with piecemeal necrosis was not
detectable in parenchyma of the TGF-β1 transgenic mice. In
consideration of this result, we speculate that the space
caused by liver necrosis in parenchyma is necessary to
induce liver fibrosis.
Furthermore, we administered Con A into female BALB/c
mice until the 12th injection to examine whether Con A-
induced liver fibrosis is progressive. We found that liver

fibrosis in the parenchyma was also detectable 1 week after
either the eighth or 12th Con A injection by Azan stain and
massive liver necrosis was detectable. However, we could
not find any apparent difference in the degree of liver fibrosis
among the groups 1 week after the fourth, eighth and 12th
Con A injection.
We also observed untreated mice after the fourth Con A
injection. Interestingly, at 3 weeks after the fourth injection
liver fibrosis in the parenchyma improved. We speculate these
results are caused by the regenerative capacity of mice
hepatocytes and the decomposing capacity of liver fibrosis.
At present the mechanism of these results has not been
defined and we need to investigate this hypothesis using this
mice model.
In conclusion, the simultaneous presence of both repeated
severe liver damage and up-regulation of TGF-β synthesis
are essential to induce hepatic fibrosis in this model.
Acknowledgements
The authors thank Dr Robert D. Schreiber (Department of Pathology,
Washington University School of Medicine, St Louis, MO) for mAb and
Dr Masanao Saio (Second Department of Pathology, Gifu University
School of Medicine, Gifu, Japan) for technical advice.
Abbreviations
aFGF acidic fibroblast growth factor
bFGF basic fibroblast growth factor
Con A concanavalin A
CTL cytotoxic T lymphocyte
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCC hepatocellular carcinoma
HGF hepatocyte growth factor
sALT serum alanine aminotransferase
TGF transforming growth factor
TNF tumor necrosis factor
References
1 Friedman, S. L. 1993. The cellular basis of hepatic fibrosis. N.
Engl. J. Med. 328:1828.
2 Milani, S., Herbst, H., Schuppan, D., Hahn, E. G. and Stein, H.
1989. In situ hybridization for procollagen types I, III and IV
mRNA in normal and fibrotic rat liver: evidence for predominant
expression in nonparenchymal liver cells. Hepatology 10:84.
3 Milani, S., Herbst, H., Schuppan, D., Kim, K. Y., Riecken, E. O.
and Stein, H. 1990. Procollagen expression by nonparenchymal
rat liver cells in experimental biliary fibrosis. Gastroenterology
98:175.
4 Rubin, E., Hutterer, F. and Popper, H. 1968. Experimental hepatic
fibrosis without hepatocellular regeneration. Am. J. Pathol. 52:111.
5 Moriyama, T., Guilhot, S., Klopchin, K., Moss, B., Pinkert, C. A.,
Palmiter, R. D., Brinster, R. L., Kanagawa, O. and Chisari, F. V.
1990. Immunobiology and pathogenesis of hepatocellular injury
in hepatitis B virus transgenic mice. Science 248:361.
6 Ando, K., Moriyama, T., Guidotti, L. G., Wirth, S., Schreiber, R.
D., Schlicht, H. J., Huang, S. and Chisari, F. V. 1993. Mechanisms
of class I restricted immunopathology. A transgenic mouse model
of fulminant hepatitis. J. Exp. Med. 178:1541.
7 Ando, K., Guidotti, L. G., Wirth, S., Ishikawa, T., Missare, G.,
Moriyama, T., Schreiber. R. D., Schlicht, H. J., Huang. S. and
Chisari, F. V. 1994. Class I-restricted cytotoxic T lymphocytes are
directly cytopathic for their target cells in vivo. J. Immunol.
152:3245.
8 Penna, A., Chisari, F. V., Bertoletti, A., Missale, G., Fowler, P.,
Mechanisms of Con A-induced hepatic fibrosis 1499
Giuberti, T., Fiaccadori, F. and Ferrari, C. 1991. Cytotoxic T
lymphocytes recognize an HLA-A2-restricted epitope within the
hepatitis B virus nucleocapsid antigen. J. Exp. Med. 174:1565.
9 Missare, G., Redeker, A., Person, J., Fowler, P., Guilhot, S.,
Schlicht, H. J., Ferrari, C. and Chisari, F. V. 1993. HLA-A31-
and HLA-Aw68-restricted cytotoxic T cell responses to a single
hepatitis B virus nucleocapsid epitope during acute viral hepatitis.
J. Exp. Med. 177:751.
10 Nayersina, R., Fowler, P., Guilhot, S., Missare, G., Cerny, A.,
Schlicht, H. J., Vitiello, A., Chesnut, R., Person, J. L., Redeker, A. G.
and Chisari, F. V. 1993. HLA A2 restricted cytotoxic T lymphocyte
responses to multiple hepatitis B surface antigen epitopes during
hepatitis B virus infection. J. Immunol. 150:4659.
11 Bertoletti, A., Chisari, F. V., Penna, A., Guilhot, S., Galati, L.,
Missale, G., Fowler, P., Schlicht, H. J., Vitiello, A., Chesnut, R. C.,
Fiaccadori, F. and Ferrari, C. 1993. Definition of a minimal optimal
cytotoxic T cell epitope within the hepatitis B virus nucleocapsid
protein. J. Virol. 67:2376.
12 Guidotti, L. G., Ando, K., Hobbs, M. V., Ishikawa, T., Runkel, L.,
Schreiber, R. D. and Chisari, F. V. 1994. Cytotoxic T lymphocytes
inhibit hepatitis B virus gene expression by a noncytolytic
mechanism in transgenic mice. Proc. Natl Acad. Sci. USA.
91:3764.
13 Guidotti, L. G., Ishikawa, T., Hobbs, M. V., Matzke, B., Schreiber,
R. D. and Chisari, F. V. 1996. Intracellular inactivation of the
hepatitis B virus by cytotoxic T lymphocytes. Immunity 4:25.
14 Toyonaga, T., Hino, O., Sugai, O., Wakasugi, S., Abe, K., Shichiri,
M. and Yamamura, K. 1994. Chronic active hepatitis in transgenic
mice expressing interferon-γ in the liver. Proc. Natl Acad. Sci.
USA 91:614.
15 Gantner, F., Leist, M., Lohse, A. W., Germann, C. G. and Tiegs,
G. 1995. Concanavalin A-induced T cell-mediated hepatic injury
in mice: the role of tumor necrosis factor. Hepatology 21:190.
16 Mizuhara, H., Uno, M., Seki, N., Yamashita, M., Yamaoka, M.,
Ogawa, T., Kaneda, K., Fujii, T., Senoh, H. and Fujiwara, H. 1996.
Critical involvement of interferon gamma in the pathogenesis of
T-cell activation-associated hepatitis and regulatory mechanisms
of interleukin-6 for the manifestations of hepatitis. Hepatology
23:1608.
17 Mead, J. E. and Fausto, N. 1989. Transforming growth factor α
may be a physiological regulator of liver regeneration by means
of an autocrine mechanism. Proc. Natl Acad. Sci. USA 86:1558.
18 Vaughan, T. J., Pascall, J. C. and Brown, K. D. 1992. Nucleotide
sequence and tissue distribution of mouse transforming growth
factor-α. Biochim. Biophys. Acta 1132:322.
19 Nakamura, T., Nawa, K. and Ichihara, A. 1984. Partial purification
and characterization of hepatocyte growth factor from serum of
hepatectomized rats. Biochem. Biophys. Res. Commun.
122:1450.
20 Russell, W. E., McGowan, J. A. and Bucher, N. L. R. 1984. Partial
characterization of hepatocyte growth factor from rat platelets. J.
Cell Physiol. 119:183.
21 Michalopoulos, G. K. and Zanrnegar, R. 1992. Hepatocyte growth
factor. Hepatology 15:149.
22 Ishiki, Y., Ohnishi, H., Muto, Y., Matsumoto, K. and Nakamura,
T. 1992. Direct evidence that hepatocyte growth factor is a
hepatotrophic factor for liver regeneration and for potent anti-
hepatitis action in vivo. Hepatology 16:1227.
23 Castilla, A., Prieo, J. and Fausto, N. 1991. Transforming growth
factors β1 and α in chronic liver disease. N. Engl. J. Med. 324:933.
24 Biagini, G. and Ballardini, G. 1989. Liver fibrosis and extracellular
matrix. J. Hepatol. 8:115.
25 Tiegs, G., Hentschel, J. and Wendel, A. 1992. A T cell-dependent
experimental liver injury in mice induced by concanavalin A. J.
Clin. Invest. 90:196.
26 Mizuhara, H., O’Neill, E., Seki, N., Ogawa, T., Kusunoki, C.,
Otsuka, K., Satoh, S., Miwa, M., Senoh, H. and Fujiwara, H. 1994.
T cell activation-associated hepatic injury: mediation by tumor
necrosis factors and protection by interleukin-6. J. Exp. Med.
179:1529.
27 Sporn, M. B. and Roberts, A. B. 1992. Transforming growth factor
β: recent progress and new challenges. J. Cell Biol. 119:1017.
28 Okuda, S., Languino, L. R., Ruoslathi, E. and Border, W. A.
1500 Mechanisms of Con A-induced hepatic fibrosis
1990. Elevated expression of transforming growth factor β and
proteoglycan production in experimental glomerulonephritis.
J. Clin. Invest. 86:453.
29 Boekelmann, T. J., Limper, A. H., Colby, T. V. and McDonald,
J. A. 1991. Transforming growth factor-β1 present at sites of
extracellular matrix gene expression in human pulmonary fibrosis.
Proc. Natl Acad. Sci. USA 88:6642.
30 Czaja, M J., Weiner, F. R., Flanders, K. C., Giambrone, M. A.,
Wind, R., Biempica, L. and Zem, M. A. 1989. In vitro and in vivo
association of transforming growth factor-β1 with hepatic fibrosis.
J. Cell Biol. 108:2477.
31 Nakatsukasa, H., Nagy, P., Everts, R. P., Hsia, C. C., Marsden,
E., Thorgeirsson, S. S. 1990. Cellular distribution of transforming
growth factor β1 and procollagen types I, III and IV transcripts in
carbon tetrachloride-induced rat liver fibrosis. J. Clin. Invest.
85:1833.
32 Shah, M., Foreman, D. M. and Ferguson, N. W. J. 1992. Control of
scarring in adult wounds by neutralising antibody to transforming
growth factor β. Lancet 329:213.
33 Border, W. A., Okuda, S., Languino, L. R., Sporn, M. B. and
Ruoslahti, E. 1990. Suppression of experimental
glomerulonephritis by antiserum against transforming growth
factor β1. Nature 346:371.
34 David, E. C., Comerford, S. A. and Hammer, R. E. 1997. Hepatic
fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome
in PEPCK-TGF-β1 transgenic mice. J. Clin. Invest. 100:2697.