1 Laboratory of Hepatobiology and Toxicology, Department of Pharmacology and Departments of 2 Surgery and 3 Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; 4 Department of Physiology and Pharmacology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098; and 5 Bayer Pharmaceuticals, D-42285 Wuppertal, Germany
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ABSTRACT |
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The role of
Kupffer cells in CCl4-induced fibrosis was investigated in
vivo. Male Wistar rats were treated with phenobarbital and
CCl4 for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl3) or were
fed glycine, which inactivates Kupffer cells. After CCl4
alone, the fibrosis score was 3.0 ± 0.1 and collagen protein
and mRNA expression were elevated, but GdCl3 or glycine
blunted these parameters. Glycine did not alter cytochrome
P-450 2E1, making it unlikely that glycine affects
CCl4 metabolism. Treatment with GdCl3
or glycine prevented CCl4-induced increases in transforming
growth factor (TGF)-1 protein levels and expression.
CCl4 treatment increased
-smooth muscle actin staining
(score 3.0 ± 0.2), whereas treatment with GdCl3 and
glycine during CCl4 exposure blocked this effect (1.2 ± 0.5); there was no staining with glycine treatment. These results
support previous in vitro data and demonstrate that treatment of rats
with the selective Kupffer cell toxicant GdCl3 prevents
stellate cell activation and the development of fibrosis.
Kupffer cells; transforming growth factor-; rat;
-smooth
muscle actin;
1(I) collagen
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INTRODUCTION |
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IT HAS LONG BEEN
HYPOTHESIZED that tissue inflammation plays a critical role in
liver pathology via induction of cellular injury. In fact, the
infiltration of mononuclear phagocytes into the liver has been shown to
correlate with the severity of alcoholic cirrhosis in humans
(25). Moreover, proinflammatory cytokines such as
interleukin-6 have been linked to fibrogenesis (29), and
the anti-inflammatory cytokine interleukin-10 is believed to inhibit
fibrosis (40). Kupffer cells are the resident macrophages of the liver, which, upon activation, release toxic cytokines and free
radicals that participate in alcoholic liver injury (6, 18,
22). Adachi et al. (1) demonstrated that
destruction of Kupffer cells with gadolinium chloride
(GdCl3) during chronic exposure to alcohol blocked early
alcoholic liver injury. Moreover, in vitro experiments have
demonstrated that Kupffer cells isolated from rats exposed to alcohol
or CCl4 produce cytokines such as platelet-derived growth
factor (PDGF) and transforming growth factor (TGF)-1 that stimulate
proliferation and production of collagen in stellate cells (7,
12, 16, 26, 38, 48). Therefore, it is likely that Kupffer cells
play an important role in the development of fibrosis.
The amino acid glycine has been used in several recent studies to prevent various forms of liver injury. For example, glycine minimized endothelial cell death due to reperfusion injury (49), improved graft function, and increased survival after orthotopic liver transplantation (3). In a rat model of endotoxin shock, dietary supplementation with glycine blunted liver and lung injury and improved survival (19). Furthermore, dietary glycine prevented necrosis and inflammation that developed early during chronic intragastric ethanol administration (17). Feeding a glycine-rich diet to rats after 4 wk of alcohol exposure also significantly enhanced the rate of recovery from early alcohol-induced liver injury (47). Therefore, dietary supplementation with glycine may be an effective therapy against liver damage, including injury caused by chronic exposure to alcohol. The mechanism of protection against injury afforded by glycine most likely involves the inactivation of Kupffer cells (19). Recent work (19) demonstrated that glycine prevents increases in intracellular calcium and cytokine production in isolated Kupffer cells exposed to endotoxin. Inhibition of calcium signaling is most likely due to hyperpolarization of the cell membrane via activation of a glycine-gated chloride channel (20). However, the effects of glycine on fibrosis have not been investigated.
The intragastric model of long-term ethanol feeding developed by Tsukamoto and French produces liver injury that closely resembles the pathology observed in human alcoholics (43); however, in rats, long periods of time and supplementation with carbonyl iron are necessary to produce consistent cirrhotic lesions with alcohol (41). In contrast, fibrosis models based on the use of chemicals such as CCl4 provide an efficient way of producing cirrhosis over short periods of time. For example, Proctor and Chatamra (32) developed a model using CCl4 and phenobarbital that produces standardized micronodular cirrhosis in 75% of rats within 8-10 wk. In the present study, this model of CCl4-induced cirrhosis was used, along with simultaneous treatment with GdCl3 or glycine, to determine whether Kupffer cells are involved in the pathogenesis of liver cirrhosis in vivo. The data presented here are consistent with the hypothesis that destruction or inactivation of Kupffer cells blunts the development of fibrosis in the liver in vivo. Preliminary accounts of this work have appeared elsewhere (45).
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MATERIALS AND METHODS |
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Animals. The protocol of animal treatment used in this study was approved by the institutional animal care and use committee. Outbred male Wistar/Han rats (225-250 g; Charles River, Raleigh, NC) were maintained on a 12:12-h light/dark cycle and given unlimited access to standard laboratory chow and water. Beginning 2 wk before treatment with CCl4, the rats were given water containing 35 mg/dl of phenobarbital. To investigate whether Kupffer cells are involved in fibrosis in vivo, the selective Kupffer cell toxicant GdCl3 (10 mg/kg) was dissolved in acidic saline and administered to rats twice each week via the tail vein beginning 1 wk before CCl4 treatment (1). Alternatively, the effects of Kupffer cell inactivation were studied by feeding standard laboratory chow supplemented with 5% casein (nitrogen control) or 5% glycine beginning 2 wk before treatment with CCl4. Data collected from rats treated with olive oil vehicle alone or with GdCl3 or glycine alone for 9 wk were combined to serve as control values.
CCl4 treatment.
The animal model for cirrhosis used in this study was described
previously by Proctor and Chatamra (32). Briefly, rats
were treated once each week, intragastrically, with CCl4 in
olive oil (0.08 ml CCl4/ml olive oil) for a total of 9 wk.
The initial amount of CCl4 administered was ~412 mg/kg
body wt. To minimize mortality, subsequent doses given were dependent
on the amount of weight gained or lost by individual rats during the
previous week (see Table 1). If the rats
gained 0-5 g, the dose of CCl4 was not changed. The
dose was increased by 32 mg after a weight gain of 6-10 g and by
64 mg if >10 g were gained. If body weight decreased 5 g, the dose
of CCl4 was decreased by 32 mg. The dose was decreased by
50% in rats that lost 6-10 g. If the rats lost >10 g, no
CCl4 was given, and treatment was resumed with the last
dose given when body weight again began to increase.
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Histology. Liver samples collected after 9 wk of CCl4 treatment were stained with trichrome, and fibrosis was scored according to the following scoring system modified from Nanji et al. (28): 1 = thickened perivenular collagen and a few thin collagen septa; 2 = thin septa with incomplete bridging between portal regions; 3 = thin septa and extensive bridging; 4 = thickened septa with complete bridging of portal regions and a nodular appearance. Collagen content was measured with a Universal Imaging (Chester, PA) analysis system as described previously (2). Briefly, trichrome-stained liver sections were analyzed with an Axioskope 50 microscope (Carl Zeiss, Thornwood, NY). An intensely labeled point was chosen to set the range of color detection for the blue trichrome stain. Collagen accumulation was calculated as the percentage of the total field at ×40 magnification that was stained blue. Fibrosis scores and collagen content in CCl4-treated rats fed chow or casein were not different; therefore, data from the control groups were combined.
Regression of fibrosis. Rats were treated with CCl4 as described in CCl4 treatment and allowed unlimited access to standard laboratory chow and drinking water containing 35 mg/dl phenobarbital. After 9 wk, CCl4 and phenobarbital treatment were discontinued. Fibrosis was scored as described in Histology with wedge biopsies of liver collected 3 days after the last injection of CCl4 to establish maximal fibrosis values. Subsequently, rats were divided into two dietary treatment groups and fed chow supplemented with 5% casein or 5% glycine. Liver biopsies were collected at 4-wk intervals for 12 wk; rats were killed 20 wk after the last injection of CCl4. Biopsied tissues were stained with trichrome to evaluate fibrosis as described above.
Measurement of 1(I) collagen mRNA.
Total RNA was harvested from liver tissue as described previously
(37). Radiolabeled riboprobes for the RNase protection assay were derived from the 375-nucleotide
PstI-AvaI fragment of rat
1(I) collagen cDNA
(39). The riboprobe for the rat GAPDH gene was generated
from the plasmid pTRI-GAPDH-Rat (Ambion, Austin, TX), which was
linearized with HindIII. Radiolabeled probes were mixed with
25 µg of total liver RNA, and the dried pellets were suspended in 30 µl of hybridization buffer (100 mM PIPES, pH 6.7, 400 mM NaCl, 2 mM
EDTA, and 80% formamide). Samples were heated at 85°C for 10 min and
then incubated at 45°C overnight. The hybridization reaction was then
performed at 37°C for 1 h in RNase buffer (300 mM NaCl, 10 mM
Tris · HCl, pH 7.6, 40 µg/ml RNase A, and 2 mg/ml RNase T1).
Subsequently, 20% SDS and proteinase K (10 mg/ml) were added, and the
reaction mixture was incubated at 37°C for an additional 15 min. The
reaction mixture was extracted with phenol and precipitated with the
addition of yeast tRNA and 100% ethanol. The samples were suspended in
formamide dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and
0.05% xylene cyanol FF) and loaded onto a standard 6% sequencing gel.
After electrophoresis, bands were visualized by autoradiography and
quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
TGF- measurement.
Frozen liver samples (500 mg) were homogenized on ice in PBS containing
1 mg/ml of aprotinin (Sigma) and 348 µg/ml of phenylmethylsulfonyl fluoride (Boehringer Mannheim). Samples were then centrifuged at 3,900 g for 10 min. For determination of total TGF-
1,
additional supernatant samples were treated with 1 N HCl to convert
latent TGF-
1 to the active form. Active and total TGF-
1 were
measured by bioassay as described previously (14).
Briefly, fibroblasts stably transfected with a TGF-
1 response
element that stimulates luciferase activity were incubated in the
presence of the supernatant isolated from liver homogenates. After
3 h, the supernatant was aspirated and luciferase lysis buffer and
luciferase substrate buffer were added. Chemiluminescence was measured
with a Hamamatsu camera and compared with signals obtained with
TGF-
1 standards.
Immunohistochemistry.
Three days after the last injection of CCl4, rats were
given a final dose of GdCl3 or saline and sections of liver
were fixed in 10% paraformaldehyde. The effect of GdCl3 on
Kupffer cells was investigated by staining 4-µm-thick liver slices
with anti-ED1 antibody (BioSource International, Camarillo, CA).
Briefly, tissue sections were deparaffinized, and endogenous peroxidase
activity was blocked by incubation of tissue in 3%
H2O2 for 10 min. Primary anti-ED1 antibody was
applied at room temperature for 20 min; rabbit IgG was used as a
negative control. ED1-positive cells were detected with a standard
immunoenzymatic staining technique (DAKO EnVision/HRP; DAKO,
Carpinteria, CA), and sections were counterstained with hematoxylin.
Additional liver slices were used for immunohistochemical detection of
-smooth muscle actin (
-SMA), a marker of stellate cell
activation, with mouse
-SMA antibody (DAKO). The extent of
-SMA
staining was scored with the following system: 1 = slight staining
confined to portal areas; 2 = moderate staining in portal areas
and light staining along collagen septa; 3 = more intense staining
with incomplete bridging between portal areas; and 4 = extensive
staining with complete bridging between portal areas.
Endotoxin measurement.
Heparinized blood samples were drawn from the tail vein at 2-wk
intervals during CCl4 treatment and from the portal vein at 9 wk, just before death. Samples were centrifuged at 150 g
for 10 min, and the plasma was stored at 80°C. Plasma samples were diluted 1:10 and heated to 75°C for 10 min to denature proteins that
interfere with the assay (10). Endotoxin was measured with a kinetic test that used a chromogenic substrate based on the Limulus amebocyte lysate assay (BioWhittaker). Pyrogen-free
water and pooled normal rat plasma were used as controls. The
concentration of endotoxin in each sample was calculated from a
standard curve prepared for each assay in plasma from untreated rats
(33). The tubes used for sample collection, storage, and
assay preparation were of borosilicate glass that was heated to 200°C
for 24 h to destroy endotoxin. Strict nonpyrogenic technique was
used for sample collection and for the assay procedure to prevent
contamination by exogenous endotoxin (33).
Measurement of p-nitrophenol metabolism. To assess cytochrome P-450 (CYP)2E1 activity, microsomes were prepared by differential centrifugation from frozen liver samples collected after 9 wk of CCl4 exposure as described previously (24). The rate of hydroxylation of the CYP2E1-specific substrate p-nitrophenol was assessed in isolated microsomes as described elsewhere (23).
Statistical analysis. Data were analyzed by one-way ANOVA or two-way ANOVA for repeated measures where appropriate, with P < 0.05 selected before the study as the level of significance. All data are presented as means ± SE of at least 4 observations/group.
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RESULTS |
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Destruction of Kupffer cells with GdCl3. After 9 wk of treatment with CCl4 or CCl4 plus GdCl3, liver sections were stained with anti-ED1 antibody and analyzed by light microscopy. There were ~31 ± 7 ED1-positive cells/50 mm2 area in livers from rats treated with CCl4. In rats treated with GdCl3, the number of ED1-positive cells was reduced by ~75%. These data are consistent with previous findings by Hardonk et al. (13) and Koop et al. (24), who showed that injection of GdCl3 destroyed ~80% of all Kupffer cells.
Effect of GdCl3 and glycine on liver pathology.
In this study, the body weight of each rat was monitored weekly. During
9 wk of treatment, comparable body weight gains were observed in rats
receiving CCl4, CCl4 plus GdCl3,
and CCl4 plus glycine (Fig.
1). Hence, the total amount of
CCl4 administered to each group was similar because of the
experimental design. Representative photomicrographs of changes in the
liver after CCl4 exposure are shown in Fig.
2. In vehicle-treated controls, only
minimal collagen staining was present; no fibrosis was detected in this
group (Fig. 2A). In CCl4-treated rats, there was
extensive collagen deposition, with septa bridging portal regions as
expected (Fig. 2B); the fibrosis score in this group was
2.9 ± 0.1. In contrast, the appearance of bridging collagen
fibers was prevented almost completely in rats treated with
CCl4 plus GdCl3 (Fig. 2C). Inactivation of Kupffer cells by dietary supplementation with glycine
also improved the histological appearance of the liver (Fig.
2D) and significantly reduced the development of fibrosis (Fig. 3).
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Effects of glycine on recovery from fibrosis.
It is possible that the protective effects of glycine may be due either
to enhanced collagen breakdown or to diminished collagen synthesis. To
determine the effect of glycine on collagen degradation, the rate of
recovery from CCl4-induced fibrosis was investigated. After
CCl4 was administered to chow-fed rats for 9 wk,
CCl4 treatment was discontinued; liver biopsies collected
at that point revealed that fibrosis was present in all rats. Rats were
then divided into two treatment groups and fed casein or glycine. The
rate of regression of fibrosis was followed as depicted in Fig.
4. Fibrosis began to decline after 4 wk
of recovery and was reduced by ~50% in both dietary groups within 20 wk. There was no difference in the rate of regression of fibrosis in
rats fed casein or glycine.
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Effect of CCl4 on plasma endotoxin levels. Endotoxin is known to activate Kupffer cells and may mediate fibrosis that results from dietary choline deficiency (4, 36). To determine whether endotoxemia occurs during CCl4-induced fibrosis, endotoxin was measured in platelet-rich plasma. Endotoxin was not detectable in plasma samples collected from the tail vein or in portal blood of oil-treated control rats. After 9 wk of CCl4 treatment, endotoxin values in portal blood were 28 ± 4 pg/ml. Plasma endotoxin levels in rats given CCl4 plus GdCl3 or CCl4 plus glycine were 24 ± 4 and 12 ± 6 pg/ml, respectively, and did not differ from rats treated with CCl4 alone.
Effect of glycine on p-nitrophenol metabolism.
Metabolic activation of CCl4 to trichloromethyl radical is
a prerequisite for hepatic injury; CYP2E1 is primarily responsible for
this process (21). It has been shown previously that
chronic administration of GdCl3 does not alter CYP2E1
activity or protein levels induced by alcohol (24). To
determine if the observed protective effects of glycine were due to
decreased hepatic metabolism of CCl4, the effect of feeding
glycine on the hydroxylation of the CYP2E1-specific substrate
p-nitrophenol was monitored after treatment with glycine or
casein (control) and CCl4 for 9 wk. The rate of microsomal
p-nitrophenol hydroxylation after CCl4 exposure
was ~0.7 ± 0.5 nmol · mg1 · min
1 and was
not different from rates measured in vehicle controls. Furthermore,
feeding rats glycine during CCl4 administration did not
significantly alter microsomal CYP2E1 activity (0.8 ± 0.3 nmol · mg
1 · min
1).
Effect of GdCl3 and glycine on collagen mRNA levels.
To assess the effects of Kupffer cell destruction or inactivation on
the rate of type I collagen synthesis, 1(I) collagen mRNA was
quantified with an RNase protection assay. A representative assay is
shown in Fig. 5. After 9 wk of
CCl4 exposure in rats fed chow,
1(I) collagen
steady-state mRNA levels were increased fivefold. Treatment of rats
with GdCl3 during CCl4 exposure blunted the
increase in
1(I) collagen mRNA expression significantly, by ~70%.
Glycine also blunted the induction of
1(I) collagen mRNA expression
due to CCl4 exposure by ~90%. Thus destruction or
inhibition of Kupffer cell function markedly diminished new collagen
synthesis in vivo.
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TGF- protein levels.
Because TGF-
is involved in fibrosis, the effects of Kupffer cell
destruction or inactivation on hepatic TGF-
1 protein levels was
investigated. Exposure of rats to CCl4 significantly
increased total levels of TGF-
1 (active + latent) in frozen
liver samples from 26.4 ± 1.2 pg/ml of liver homogenate in
oil-treated controls to 55.9 ± 5.3 pg/ml. In contrast, TGF-
1
expression in livers from rats treated with GdCl3 during
CCl4 exposure was not increased and was 28.2 ± 3.2 pg/ml of liver homogenate. Glycine was most effective and actually
reduced TGF-
1 expression (2.8 ± 0.7 pg/ml).
Effects of glycine and GdCl3 on -SMA expression.
It is well known that CCl4 causes stellate cells to undergo
phenotypic transformation to myofibroblast-like cells (44,
48); expression of
-SMA is characteristic of the new
phenotype. Therefore, the effects of CCl4, glycine, and
GdCl3 on stellate cell
-SMA expression were measured
with immunohistochemistry.
-SMA was not detectable in liver sections
from oil-treated control rats (Fig.
6A). As expected,
CCl4 treatment resulted in extensive
-SMA staining in
portal areas, and the staining appeared to extend along collagen septa
bridging portal areas; the average score for
-SMA staining in this
group was 3.0 ± 0.2 (Fig. 6B). Treatment of rats with
GdCl3 during CCl4 exposure largely blocked
expression of
-SMA and resulted in a score of 1.2 ± 0.5 (Fig.
6C). Staining was prevented completely in rats fed a
glycine-rich diet (Fig. 6D).
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DISCUSSION |
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Effect of GdCl3 and glycine on Kupffer cells. Previous experiments have shown that intravenous administration of GdCl3 depletes Kupffer cells. For example, by using electron microscopy, Hardonk et al. (13) demonstrated that large Kupffer cells were no longer present 24 h after GdCl3 treatment. Recently, Koop et al. (24) showed that GdCl3 eliminated ~80% of a Kupffer cell-specific lectin ("Kupffer cell receptor"). Consistent with these findings, a 75% decrease in the number of ED1-positive Kupffer cells was observed after 9 wk of treatment with GdCl3 in the present study.
Chronic treatment with GdCl3 (24), or feeding glycine as performed in the present study, did not alter CYP2E1 activity; therefore, it is unlikely that these agents interfere with the bioactivation of CCl4 to the toxic trichloromethyl metabolite. In a previous study, glycine prevented endotoxin-induced Kupffer cell activation and cytokine production by stimulating chloride influx and hyperpolarizing the cell membrane (20). Therefore, the protective effects of glycine against fibrosis most likely result from diminished Kupffer cell activity; however, a direct effect of glycine on stellate cell type I collagen production cannot be ruled out (see below).GdCl3 and dietary glycine diminish hepatic fibrosis.
Fibrosis is a complex pathological process. In early stages,
inflammation and necrosis occur, which may initiate hepatocyte regeneration and repair. Subsequently, there is increased accumulation of matrix proteins, and alterations in the normal architecture of the
hepatic lobule occur. Impairment of metalloproteases, such as transin,
that degrade the extracellular matrix is believed to contribute to
fibrosis that results from CCl4 exposure (15). In the present study, injury ranging from fibrosis to cirrhosis was
observed (Fig. 2), confirming other work (32, 35, 45). Treatment of rats with GdCl3 or glycine during
CCl4 exposure diminished collagen accumulation (Fig. 2,
C and D) and markedly reduced the appearance of the bridging septa of matrix proteins between portal areas that is characteristic of advanced fibrosis (Figs. 2 and 3). On
the other hand, feeding glycine did not influence the rate of
regression of fibrosis (Fig. 4), consistent with the hypothesis that
glycine does not enhance collagen degradation and most likely does not
effect metalloprotease activity. Therefore, it is likely that the
protection against fibrosis observed in this study was due to decreased
synthesis of matrix proteins. Indeed, the increase in 1(I) collagen
mRNA expression caused by CCl4 was largely blocked by
GdCl3 and glycine (Fig. 5). These results demonstrate that removal or inactivation of Kupffer cells in vivo has profound inhibitory effects on the pathogenesis of fibrosis.
Possible mechanism of Kupffer cell involvement in
CCl4-induced hepatic fibrosis.
It is well known that Kupffer cells release toxic free radicals and
cytokines (6), and in vitro studies have demonstrated that
Kupffer cells produce factors that activate stellate cells (48). As illustrated in Fig.
7, two important cytokines are PDGF and
TGF-1; there is evidence that they both play important roles in the
proliferation and enhanced production of matrix proteins by stellate
cells (8, 26). Although stellate cells are capable of
producing TGF-
1 (27), recent studies indicate that
increased expression of TGF-
1 mRNA in Kupffer cells isolated from
rats with alcoholic fibrosis precedes expression in stellate cells (42). PDGF stimulates the release of retinol, an early
event in stellate cell activation (9). Furthermore, PDGF
receptor expression correlates with cell proliferation and collagen
deposition in the early stages of CCl4-induced liver injury
(46). Another profibrogenic cytokine, interleukin-6, has
also been implicated in stellate cell activation (11, 42).
Collagen production by cultured stellate cells has been shown to be
stimulated by the addition of interleukin-6, and in vivo experiments
have demonstrated that interleukin-6 mRNA levels in Kupffer cells are
markedly increased, coincident with stellate cell transformation to
myofibroblast-like cells.
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Conclusions.
As illustrated in Fig. 7, it is hypothesized that chronic
CCl4 exposure activates Kupffer cells, possibly via the
release of radicals or by causing mild endotoxemia. The present study demonstrates that destruction of Kupffer cells with GdCl3,
as well as Kupffer cell inactivation with dietary glycine, diminishes stellate cell -SMA expression and collagen production, most likely by preventing the release of profibrogenic cytokines from Kupffer cells. Alternatively, previous studies (25, 29, 40)
suggested that early inflammation contributes to the later development
of fibrosis. Because GdCl3 has been shown to prevent tissue
injury and inflammation resulting from acute CCl4 exposure
(7), it is also possible that Kupffer cell
destruction diminishes fibrosis by blunting early hepatic necrosis and
inflammation. Whereas conditioned medium and coculture experiments
predict a role for Kupffer cells in fibrogenesis (12, 26, 38,
48), this study provides direct in vivo evidence that Kupffer
cells are involved in this pathology. Thus agents that selectively
block Kupffer cell activation may provide effective therapy against the
progression of fibrosis in humans.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. U. Bradford, 1124A Mary Ellen Jones Bldg., Campus Box #7365, Chapel Hill, NC 27599 (E-mail: beub{at}med.unc.edu).
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. Section 1734 solely to indicate this fact.
Received 2 December 2000; accepted in final form 6 March 2001.
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