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

since the mouse liver has placed for 15 min on ice. polymerase (Boehringer). Thus. TGT TTG GGT CAG TTG-39 for HGF respectively. 59-CGG GGG after the i. To eosin. 1 µg of total Methods RNA was denatured by heating for 10 min at 70°C and added to a mixture containing 2 µl of oligo-dT (0. Total RNA (1 µg) was reverse transcribed into cDNA by using AMV reverse transcriptase (Boehringer. West Chester. Thermal cycle conditions were 94°C Serum samples from individual mice were obtained from the for 45 s for denaturing. 60°C for 45 s for annealing and 72°C for 2 min for extension. TGF-β1 and G3PDH.p. Japan). Total RNA contents of the be difficult to establish a liver fibrosis model after hepatocellu. 0. ensure that contaminating DNA had not been amplified. TGF-α. since adoptively transferred HBsAg-specific CTL hepatic RNA was isolated for the analysis of cytokine mRNA abolish the HBV gene and antigen expression in the liver expression at the indicated time points after Con A injection. Mannheim. Hepatocellular injury was monitored biochemically dilutions of cDNA were amplified for 20. it has been homogenate. TNF-α and TGF-β1 mRNAs was performed by . commercially available ELISA kits (Gibco/BRL. 28. Purity of RNA was determined from We now report that concanavalin A (Con A)-induced hepat. it has phenol–chloroform method using RNAzol (Tel-Test. MO) was dissolved in pyrogen-free µg of cDNA. the RNA pellet was washed once with ice-cold 75% cytes. in which initial necroin. KCl (50 mmol/l). acidic fibroblast MD and Genzyme. Each cDNA synthesis was performed for 1 h at 42°C and stopped by incubation for 10 min at 98°C. pH 8. of IFN-γ. 59-TTC CCA CCA GGC CAC TTC A-39 generously provided by Dr Robert D. a mouse model of hepatic fibrosis following immunolo. 12. 2 Con A (Sigma. PCR was performed Disease model in a volume of 50 µl containing 400 pmol/l of each primer. Tris–HCl (10 mmol/l. sense primers were 59-TGC CCA GAT TCC CAC ACT-39 and α in the fibrosis process. 23. TX).13). mice were sacrificed 1 and to permit semiquantitative analysis of signal strength. After centrifugation at 12.3 mg/mouse once a week. To evaluate the role of IFN-γ and TNF. Cambridge. Briefly.5 mmol/l).2% agarose gels and stained with ethidium bromide. 1 µl of dNTP Female BALB/c mice (7–10 weeks old. The PCR products were electrophoresed on Sections of 5 µm thickness were stained with hematoxylin & 1. Liver tissues were fixed the last cycle of amplification. by measuring serum alanine aminotransferase (sALT) activity. Sense and anti-sense primers were based on Clontech The serum levels of IFN-γ and TNF-α were monitored using (Palo Alto. The detect. The number of amplification cycles funds oculi vein at indicated time points after each Con A was 28 for each set of primers. 6 µl of 5-fold concentrated reverse transcriptase Mice buffer.3). 15 min. The sense and anti- 35 pg/ml respectively. PBS (Sigma) at a concentration of 1 mg/ml and i. 34. MA respectively).1 mol/l). Schreiber (Department and 59-TGC CCA GTT CGT TTC AGT GC-39 for bFGF.12 CCA CTT CTT GAG GA-39 and 59-ACC GGG AGG GGC AGA specific for murine IFN-γ and TNF-α respectively. The samples were shaken vigorously for 15 s reported that liver-specific expression of IFN-γ in transgenic and incubated for 5 min on ice. Friendswood. TNF-α. CA) Amplimer sets for mouse IFN-γ.CTC GGA Louis. mixed with an equal volume of isopropanol and in the portal areas (14). tissue samples were homogenized although it is speculated that continuous hepatocellular injury with RNAzol and 0. weight 25–30 g) were (Boehringer. and of Pathology. Frozen liver tissue was mechanically pulverized and total However. dried gically induced hepatocellular injury has not yet been for 10 min and dissolved in diethylpyrocarbonate (Sigma)- produced. itis develops hepatic fibrosis after repeated liver injury and the mechanisms are mainly based on the up-regulation of RT-PCR analysis transforming growth factor (TGF)-β1 expression in the liver. obtained from Japan SLC (Shizuoka. St Louis. absorbance at 260 nm.v.2 ml chloroform was added to 2 ml of may induce liver fibrosis. injection of hamster mAb H22 and TN3 19. injection. According to these results. it has been thought to treated RNase-free solution. growth factor (aFGF).1492 Mechanisms of Con A-induced hepatic fibrosis hepatitis B (8–11) and thus the HBsAg transgenic mouse RNA preparation model may have essential similarities to human hepatitis. Germany) and the cDNAs were amplified by PCR. 7 min at 72°C. For each set of primers. been difficult to establish liver fibrosis using this mouse model. ethanol with vortexing. Quantitation PA) was used as a control antibody. 32. samples were estimated spectrophotometrically by lar injury mediated by immunological mechanisms. In addition.5 mg/ml) (Boehringer). 30. Azan or silver for light-microscopic evaluation. 36 and 38 cycles to define optimal conditions for linearity For the histopathological evaluation. 10 mmol/l) and 1 µl of reverse transcriptase. 2 µl of DTT (Boehringer. After week after the final injection of Con A. basic FGF (bFGF) and hepatocyte able lower limits of sensitivity of the kits were 125 and growth factor (HGF) were investigated. St 59-CCG AGG CCA TGG TGC TAC A-39 and 59. Washington University School of Medicine. 25. Purified hamster IgG (Organon. injected MgCl2 (1. Con A was administered at 24 h 59-TGG ATC AGC ACA CAG GTG for TGF-α. With another approach.000 g for 15 min. In brief. the samples were incubated for in 10% neutral-buffered formalin and embedded in paraffin. centrifuged at 7500 g for 5 min. PCR without reverse transcriptase was simultaneously demon- Serum levels of IFN-γ and tumor necrosis factor (TNF)-α strated. and then centrifuged at mice causes chronic active hepatitis.2 mmol/l) and 2 U of Taq DNA into BALB/c mice at a dose of 0. dNTP (0. Gaithersburg. The aqueous phase was transferred to flammatory changes are followed by lymphoid cell infiltration a fresh tube. MO). the 260/280 absorbance ratio. However. which were AAC AA-39 for aFGF. (12.000 g for a remarkable regenerative capacity for individual hepato. a 4 week interval of CTL administration is required to Total RNAs were isolated by the guanidinium isothiocyanate– induce an equal level of liver injury in this model.

v. Twenty-four hours after first Con A injection. immunization with anti-IFN-γ mAb and anti-TNF-α mAb in this model. 16.v. Sera and liver tissues observed at this period. as compared with the Con A injection alone.v. as shown in Fig. critical roles in Con A-induced liver injury (15. 4(A and B). aFGF and TGF-β1. purchased from Genzyme (Cambridge. 1A). 1. No hepatocellular necrosis and were collected at several time points after the administration hepatic fibrosis were found 1 week after the second Con A of Con A and anti-TGF-β mAb for analysis of both hepatic injection (Fig. Each data and error bar bFGF. As shown in Fig. 5(A). 3(A). fibrosis. Cytokine gene expression in liver after Con A administration Fig. we investigated the protective effects of passive With weekly administrations of Con A. However. As shown in Fig. Hepatocellular injury was monitored by analyzing sALT levels 0. 2 fourth injection. 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. Statistical analysis Role of IFN-γ and TNF-α Values were expressed as mean 6 SD. Hepatocellular injury during early phase after Con A injection To determine whether cytokines contribute to the hepatic (A). 24. fibrosis. Mechanisms of Con A-induced hepatic fibrosis 1493 competitive PCR using the PCR Mimic Protocol (Clontech). Similarly.3 mg/ mouse. 3B and C) at 1 week after the fourth Con A injection. bridging mRNA expressions and serum levels of IFN-γ and TNF-α.p. Differences between Since it has been reported that both IFN-γ and TNF-α play experimental and control groups were analyzed by the Krus. MA). 8. Anti-IFN-γ mAb or anti-TNF-α mAb (150 µg/mouse) were administrated i. 24 h after the first injection. 2B). No evidence of hepatic fibrosis was i. liver tissues were derived from Role of TGF-β1 in hepatic fibrosis mice before Con A injection. sALT levels were analyzed 24 h after each weekly Con A mRNA expression of TGF-α. was hepatocellular necrosis had become widespread throughout simultaneously administered (200 µg/mouse/each injection) the lobule (Fig. injected with Con A at a dose of 0. reported to affect tissue represent the mean of results in five mice and the SD respectively. TNF-α and TGF-β1 competitor primers yielding product injection. 2(D). 500 and 390 bp respectively were used in each reaction. 2C). injected Con A at a dose of 0. 24 h before each Con A injection. and silver staining (inset). we analyzed the mRNA expression levels of TGF-α. aFGF was negatively regulated. To evaluate the role of TGF-β in the development of liver 1 week after the second injection and 1 week after the fibrosis. (B). HGF. an 87% reduction in the peak of TNF-α was observed after Con A injection with anti- IFN-γ mAb. Each bar and error bar represent the mean of results in after Con A injection (peak 5 4. In contrast. we investi- kal–Wallis test followed by Scheff’s F-test. 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. Serum levels of IFN-γ gradually increased Results and reached a peak 12 h after a single Con A injection (4328 The evaluation of hepatocellular injury induced by repetitive 6 1109 pg/ml). The In order to reveal the role of IFN-γ and TNF-α in hepatic levels then rapidly decreased within 48 h (490 6 130 IU/l). Serum levels of TNF-α strikingly increased Con A injections and reached a peak within 2 h (1122 6 105 pg/ml) (data not shown).3 mg/mouse. seven mice and the SEM respectively. G3PDH competitor primers was Histopathological analysis of hepatic fibrosis constructed using a PCR Mimic construction kit (Clontech). the monoclonal neutralizing antibody to TGF-β1. sizes of 500. Quantitative RT-PCR performed using liver tissues After Con A was i. and 3. the levels of IFN-γ showed a 84% reduction in the peak after Con A injection with anti-TNF-α mAb. sALT levels began to rise at 4 h (434 6 injection (data not shown). As shown Fig. gated changes in both serum levels and hepatic mRNA expression of IFN-γ and TNF-α at several time points after Con A injection.16). 4. BALB/c mice were i. fibrosis in the parenchyma with hepatocellular necrosis was Histopathological examination was also performed using the detectable 1 week after the fourth Con A injection by Azan liver tissues. as compared with the Con A injection alone. Furthermore. Histopathological examination similarly revealed inhibition of hepatocellular injury and hepatic fibrosis (Fig. 1(B). bFGF and HGF was up-regulated injection. fibrogenesis (17–22) by either semiquantitative or competitive Effect of repeated administration of Con A on hepatocellular injury RT-PCR after Con A administration. 138 IU/l) and reached a peak at 24 h (4840 6 1194 IU/l). To evaluate whether the inhibitory effects were due to independent pathways. striking increases of sALT were observed 24 h after each IFN-γ. as shown in Fig.3 mg/mouse. BALB/c mice were i.p. Competitive . 36 and 48 h after Con A injection. reaching peaks within 4 h after Con A time points (Fig. at the time of each Con A injection. To assess the evolution of pathological changes in livers due to weekly Con A injections. 24 and 24 h respectively). injected into BALB/c mice at a dose of also revealed up-regulation of hepatic mRNA expression of 0. sALT activities were measured at indicated both IFN-γ and TNF-α.

venting hepatic fibrosis in this model. induced hepatic fibrosis 5B). Original magnification 3200. we focused on the role of TGF-β1 of IFN-γ was enhanced.v. 7(A). serum levels and Prevention of Con A-induced hepatic fibrosis by anti-TGF. that of TNF-α was not among the cytokines studied above in the development of enhanced by the simultaneous treatment of anti-TGF-β mAb hepatic fibrosis in this mouse model. 6C). hepatic mRNA expression chronic hepatitis (23). inflammatory liver disease. after each Con A injection either with or without administration of anti-TGF-β mAb from weeks 1 to 4. 7B). Azan staining for (C). Reflecting these results.1494 Mechanisms of Con A-induced hepatic fibrosis Fig. Relationship between TGF-β and IFN-γ or TNF-α in Con A- regulated and reached a peak 48 h after Con A injection (Fig. 6A). hepatic mRNA expression of IFN-γ and TNF-α were analyzed β mAb after Con A injection.3 mg/mouse once per week. Histopathological evaluation of the livers of Con A-treated mice. Treatment with either anti-IFN-γ or anti-TNF-α mAb As a first step to determine whether TGF-β1 affects the liver did not affect the expression levels of TGF-β1 mRNA in liver injury induced by Con A. we monitored sALT activities 24 h after Con A injection (Fig. 24 h after the first injection (B). Histopathological characteristics of liver without Con A injection (A). As shown in Fig. 2. There was no significant difference in sALT levels between the two groups at any time Discussion points (Fig. 1 week after the second injection (C) and 1 week after the fourth injection (D). Although the changes in hepatic . These results indicated that most of the cytokine’s mRNAs To determine the relationships between the cytokines pre- except for aFGF were up-regulated after Con A injection. serum levels of Since it has been recently reported that the hepatic expression IFN-γ. 7B). with or without simultaneous anti-TGF- β mAb treatment. Note the presence of hepatic fibrosis (arrowheads) in (D). Representative figures derived from analyses of four mice are shown in each time point. were significantly increased by the of TGF-β1 mRNA is markedly up-regulated in patients with simultaneous treatment of anti-TGF-β mAb 4 h after Con A liver cirrhosis. but not TNF-α. In contrast. as compared with both normal control and injection. (Fig. histopathological examination revealed that the administration of anti-TGF-β1 mAb markedly Fibrosis is an important component of advanced chronic inhibited hepatic fibrosis (Fig. injected with Con A at a dose of 0. RT-PCR revealed that mRNA expression of TGF-β1 was up. BALB/c mice were i. in contrast. and Azan staining and silver staining (inset) for (D). Hematoxylin & eosin staining was performed for (A) and (B).

it is accepted that the model requires T cell activation. (ii) hepatic fibrosis should be induced following continu. we established a hepatic fibrosis model mental liver injury in mice is mediated by activated T cells with repeated administrations of Con A (Fig. since the expression of HBsAg is down-regulated to be one of the potent models to induce liver fibrosis mediated by the CTL. 0.001) is significantly lower than a group of mice treated with control antibody (A). Since these results indicate As cited above. the animal model of liver injury induced by that Con A-induced hepatitis is based upon immunological HBV-specific CTL in HBV transgenic mice has similarities to mechanisms and has similarities to chronic inflammatory liver human cases (6). preferential binding of Con A in the liver. features: (i) it should be based upon immunological mechan. 24 h before weekly Con A injections. by hepatocellular injury.v. Because immunode- lying mechanisms. Representative figures derived from the analysis of three mice are shown in each group. FK506 and lished.3 mg/mouse). However. Tiegs et al.16). Con A is a lectin from jack bean and is known as a injury was consistently observed in terms of sALT elevation T lymphocyte mitogen in vitro. Azan staining was performed for (B) and (C). 1A and B) following an . 3. an appropriate animal model with essential ficient mice (SCID or athymic mice) or mice treated with similarities to patients with chronic hepatitis should be estab. liver injury cannot be continuously disease in man. Control rat-IgG. The point marked (**P . depletion experiments identified CD41 T cells as the effector ous liver injury and (iii) the inducer of hepatic fibrosis is should cells (25). Con A-induced hepatitis model was thought induced. 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. structures that occur in liver cirrhosis have been described liver injury was induced within 8 h after i. anti-IFN-γ or anti-TNF-α mAb (150 µg/mouse) were administered i.26). Ideally. Role of IFN-γ and TNF-α in hepatocellular injury and hepatic fibrosis. Each value represents the mean 6 SD of results in three mice. Furthermore. 2). It has been recently described that Con A-induced experi. Cell isms. reported that acute 24 h after each Con A injection (Fig. neutralization experiments revealed not be a direct hepatotoxin. immunosuppressive drugs such as cyclosporine. In order to elucidate the under. Hepatocellular injury was monitored by analyzing sALT levels. the animal model should have the following corticosteroids do not develop hepatitis after Con A injection. the immunopathogenesis of fibrosis in this in BALB/c mice. Hepatocellular (25. no animal model that that TNF-α and IFN-γ play critical roles in the development of satisfies these criteria has been established. In the present study. The organ specificity was attributed to the process is largely unknown. Sera and liver tissues were sampled from BALB/c mice 1 week after each weekly Con A injection (0. Original magnification 3100. However. injection of Con A in detail (24). Con A-induced hepatitis (15. Mechanisms of Con A-induced hepatic fibrosis 1495 Fig. Pretreatment with either anti-IFN-γ or anti-TNF- α mAb completely inhibited hepatocellular injury as compared with that with control rat-IgG.p.

Fig. 23. c 5 6. 5. In particular. α and HGF. cytokine involvement could be proven in this model. Control rat IgG. factors. bFGF. insulin-like growth factor. 4. Hepatic TGF-α. 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. Representative figures from the analyses of four mice are shown in each group.25310–2 and d 5 5310–2 amol/µl. The quantitation of TGF-β1 mRNA was performed by competitive RT-PCR using the PCR Mimic protocol (Clontech). 34. 36 and 38 cycles to define optimal conditions for linearity and to permit semiquantitative analysis of signal strength. 30. FGF.3 mg/mouse). TGF-β has been identified as the .3 mg/mouse) (A).5310–1. including TGF-β. Liver samples were obtained at the times shown. HGF and aFGF mRNA levels were analyzed by semiquantitive RT-PCR at indicated time points after Con A injection (0. TGF- 3A). The concentrations of competitors were as follows: a 5 5310–1. Each value represents the mean 6 SD of results in five mice.1496 Mechanisms of Con A-induced hepatic fibrosis Fig. 28.p.001) is significantly higher than a group of mice treated with either anti-TNFα mAb or anti-IFNγ mAb. TGF-β1 mRNA was assessed by competitive RT-PCR (B). The number of amplification cycles was 28 for each set of primers. 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. b 5 1. anti-IFN-γ or anti-TNF-α mAb (150 µg/mouse) were administered i. Dilutions of cDNA were amplified for 20. 25. 0. Biosynthesis and turnover of the extracellular matrix are 2A). 24 h before a single Con A injection (0. Cytokine expression in the liver after Con A administration. G3PDH was used as the internal control (A). increase in serum levels of both IFN-γ and TNF-α (Fig. Hepatocellular injury was monitored by analyzing sALT levels. 32. The point marked (**P .

Azan staining was performed for (B) and (C). regulation of either IFN-γ or TNF-α synthesis. while treatment with anti-IFN-γ. TGF-β1 activity has been successfully with either anti-IFN-γ or anti-TNF-α mAb did not affect hepatic suppressed in vivo with anti-TGF-β1 antibodies. anti-TNF-α or and TGF-β1 mRNAs was up-regulated after each Con A anti-TGF-β mAb markedly prevented hepatic fibrosis. Either bovine IgG (200 µg/mouse) or anti-TGF-β mAb (200 µg/mouse) was administered i. Mechanisms of Con A-induced hepatic fibrosis 1497 Fig. treatment with either anti- nephritis and idiopathic pulmonary fibrosis (1. treatment In two recent studies. III and IV mRNAs in the livers of enhanced by the simultaneous treatment of anti-TGF-β mAb rats with carbon tetrachloride-induced hepatic fibrosis (31).29). that of TNF-α was not mRNA. Representative figures derived from the analyses of three mice are shown in each group. 5A and B).28. simultaneous administration of separate. it is speculated that TGF-β is one (Fig. 6. bFGF (Fig. Thus. IFN-γ or anti-TNF-α mAb completely prevent hepatic injury In the present study. 7B). HGF. significant decrease in excess extracellular matrix deposition as compared with Con A injection alone. and procollagen I. Reflecting these results. Each value represents the mean 6 SD of results in three mice (A). 7A). B) or anti-TGF-β mAb (200 µg/ mouse. Since. On the other hand. Hepatocellular injury was monitored by analyzing sALT levels 24 h after each weekly Con A injection. the mechanism of prevention of hepatic It has furthermore been reported that hepatic TGF-β1 mRNA fibrosis by anti-TGF-β mAb is clearly not through the down- was enhanced in patients with chronic liver disease (23). into BALB/c mice simultaneously with each Con A injection (0. the administration (Fig.3 mg/mouse). Thus. tissue repair. Original magnification3200. observed. This result indicated that TGF-β synthesis is not .p. (Fig. C). and cell growth and reduced hepatic fibrosis and extracellular matrix formation differentiation (27). in parallel with an injection (Fig. hepatic mRNA expression also demonstrated a positive correlation between TGF-β1 of IFN-γ was enhanced. In the present study. most potent fibrogenic mediator.33). although it exerted no significant effects on hepato- of the major causes of fibrosis in hepatic cirrhosis. cellular injury. in contrast. 3A). 6B). Histopathological evaluation of the liver 1 week after the fourth Con A injection with either control bovine IgG (200 µg/mouse. Prevention of Con A-induced hepatic fibrosis by anti-TGF-β mAb. Note the marked inhibition of hepatic fibrosis in (C) as compared with that in (B). their influences are (32. 7B). Furthermore. Nakatsukasa et al. but no equivalent influence on TNF-α was increase in type I procollagen mRNA (30). resulting in a mRNA expression of TGF-β1 after Con A injection (Fig. Thus. Using an experimental model of schistosomiasis in mice. prevention was based on different mechanisms. glomerulo. similarly found an increase in the content of simultaneous treatment of anti-TGF-β mAb 4 h after Con A hepatic TGF-β1 mRNA in infected animals. expression of TGF-α. serum levels of IFN-γ were significantly increased by the Czaja et al. contributing to immune and neutralizing TGF-β mAb with each Con A injection markedly inflammatory responses.

The effects of anti-TGF-β mAb on the expression of IFN-γ and TNF-α mRNA in liver after Con A injection (B. consideration of this result. Serum levels of IFN-γ were significantly increased by the simultaneous treatment of anti-TGF-β mAb 4 h after Con A injection (**P . In injury is also not enough to induce hepatic fibrosis. Results are from the analyses of three mice in each group. attomol/µl (B).. 0.5310. In addition. 0. However.1498 Mechanisms of Con A-induced hepatic fibrosis Fig.5310–1. f 5 5310. Relationships between TGF-β. Each value represents the mean 6 SD of results in three mice (A). We found that liver . since treat. e 5 1. 5B). liver fibrosis induced liver fibrosis is progressive. such as bridging fibrosis with piecemeal necrosis was not ment with anti-TGF-β mAb prevented 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. see Fig. b 5 1. 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.05). In contrast. 7. 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). we administered Con A into female BALB/c that marked collagen deposition was shown localized to the mice until the 12th injection to examine whether Con A- sinusoids and around periportal areas. d 5 5310–2.p. g 5 1. and h 5 6. The induce liver fibrosis. IFN-γ and TNF-α in Con A-induced hepatic fibrosis. 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. repeated liver detectable in parenchyma of the TGF-β1 transgenic mice. upper panel).01). c 5 6. authors established TGF-β1 transgenic mice and indicated Furthermore.25310–2. lower panel). sufficient to develop hepatic fibrosis. (34). serum levels of TNF-α were significantly decreased 8 h after Con A injection (*P .5310–2. The concentrations of competitors were as follows: a 5 5310–1.25310–1. Liver tissues were derived from BALB/c mice 48 h after Con A injection (at a time of peak expression of TGF-β mRNA. 200 µg/mouse) at indicated time points. we speculate that the space An interesting report has recently been published regarding caused by liver necrosis in parenchyma is necessary to the correlation between TGF-β1 and liver fibrosis. data not shown).

. 1994. B.. A transgenic mouse model 26 Mizuhara. 1994. of class I restricted immunopathology. J. Biochem. 119:1017. at 3 weeks after the fourth injection Schlicht. Kusunoki. Guidotti. Redeker. Transforming growth 5 Moriyama. L. Schuppan. Hepatol. J.. 174:1565.. H. G.. S.. Immunity 4:25. J. Fowler.. CTL cytotoxic T lymphocyte Critical involvement of interferon gamma in the pathogenesis of HBsAg hepatitis B surface antigen T-cell activation-associated hepatitis and regulatory mechanisms HBV hepatitis B virus of interleukin-6 for the manifestations of hepatitis.. Kim. Vitiello. L. Proc. K. 328:1828. R... and Border. O.. USA 86:1558. J. V.. H.. Yamashita. Cell Biol. M. Huang. and Bucher. Leist.. J. Japan) for technical advice. and Fausto... Muto. expression in nonparenchymal liver cells. A. T.. Schreiber. injection. J. D. Riecken. St Louis. S. K. Partial purification References and characterization of hepatocyte growth factor from serum of 1 Friedman.. 1992.. 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