Liver Unit, Gastroenterology Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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Endogenous glucocorticoids are known to play a
role in the regulation of the inflammatory response possibly by
modulating pro- and anti-inflammatory cytokine expression. We examined
endogenous glucocorticoid secretion, hepatic damage, tumor necrosis
factor- (TNF-
), and interleukin-10 (IL-10) mRNA expression and
release in rats treated with carbon tetrachloride
(CCl4) after treatment with
vehicle or a glucocorticoid receptor antagonist (RU-486). Rats treated
with CCl4 demonstrated striking
elevations of plasma corticosterone levels. Inhibition of endogenous
glucocorticoid activity by pretreatment with the glucocorticoid
receptor antagonist RU-486 resulted in augmented
CCl4-mediated hepatotoxicity, as reflected by histology and serum transaminase levels, which were independent of alterations in serum TNF-
levels or hepatic mRNA expression. CCl4 treatment
resulted in enhanced hepatic IL-10 mRNA expression and elevated serum
IL-10 levels, which were markedly attenuated by glucocorticoid receptor
blockade. In summary, significant endogenous glucocorticoid release
occurs during acute toxic liver injury in the rat and suppresses the
inflammatory response independent of effects on TNF-
but possibly by
upregulating hepatic IL-10 production.
cytokine; inflammation; corticosterone; RU-486; carbon tetrachloride
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INTRODUCTION |
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INFLAMMATORY PROCESSES are capable of stimulating
endogenous glucocorticoid secretion by activating the
hypothalamic-pituitary-adrenal axis (13). This activation has been
postulated to occur via proinflammatory cytokines [e.g.,
interleukin-1 (IL-1), IL-6, tumor necrosis factor- (TNF-
)]
that act to stimulate all levels of the hypothalamic-pituitary-adrenal
axis (13). Glucocorticoids (corticosterone in the rat) released from
the adrenal cortex during the inflammatory process are thought to play
an important role in controlling the development and resolution of
inflammation. Specifically, glucocorticoids directly influence immune
cell function, adhesion molecule expression, and inflammatory mediator
and cytokine generation (reviewed in Ref. 6). Previous work has shown
that endogenous glucocorticoid secretion occurs during acute toxic liver injury in rats (21), but the effects of the released
glucocorticoids on hepatotoxicity and cytokine release are poorly defined.
Carbon tetrachloride (CCl4) is a
hepatotoxic compound that is used widely as an inducer of acute and
chronic experimental liver disease (35). Single-dose treatment of rats
with CCl4 induces acute liver
injury characterized by zone 3 liver cell necrosis and steatosis (35).
TNF- has been suggested as playing a role in liver cell damage due
to CCl4 (9), as well as playing a
central role in recovery from CCl4
hepatotoxicity (1, 3, 4). Furthermore, endogenous glucocorticoid
secretion has been shown to be capable of modulating TNF-
synthesis
and release in endotoxin-treated rats and mice (23, 36). Alternatively, administration of pharmacological doses of glucocorticoid appears to be
able to increase the endogenous release of the anti-inflammatory cytokine IL-10 in humans and mice (19, 30, 32). Moreover, exogenous
administration of IL-10 has been shown to protect the liver from toxic
damage induced by a number of hepatic insults (17, 18).
Therefore in this series of experiments we quantified the magnitude of
endogenous glucocorticoid secretion in rats treated acutely with
CCl4 and examined the effects of
the inhibition of endogenous glucocorticoid activity on the extent of
CCl4-induced hepatotoxicity as
well as on TNF- and IL-10 synthesis and release.
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METHODS |
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Animal model. Male Sprague-Dawley rats weighing ~200 g were obtained from Charles River (Pointe Claire, QC, Canada). All animals were housed in a light-controlled room maintained at 22°C with a 12:12-h day-night cycle and were given free access to food and water. The rats were handled regularly to avoid handling stress during experiments. All animals were treated humanely under University of Calgary Animal Care Committee guidelines.
The model of acute liver injury used was that due to CCl4 treatment (35). Rats were fasted for 12 h before and for 6 h after CCl4 (Sigma Chemical, St. Louis, MO) treatment. CCl4 (0.3 ml/200 g body wt in corn oil 1:1) or corn oil vehicle were given by gavage, rats were killed 0, 12, and 24 h later, blood samples were obtained by vena caval puncture (under halothane anesthesia) for TNF-Serum transaminase and corticosterone determinations. Plasma alanine aminotransferase (ALT) levels were determined using a commercially available kit (Sigma). Plasma corticosterone levels were determined using a sensitive RIA as described previously (29).
Plasma cytokine assays.
Plasma TNF- bioactivity was determined using a highly sensitive
assay modified from Mosmann (20). This assay determines TNF-
cytotoxicity against WEHI 164 murine fibrosarcoma cells (CRL 1751, American Type Culture Collection, Rockville, MD) in the presence of
actinomycin D and uses
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) as colorant. Briefly, 5 × 105 cells were incubated in
96-well microtiter plates at 37°C with 5%
CO2 in 50 µl RPMI 1640 medium
(GIBCO, Gaithersburg, MD) containing 10% fetal bovine serum, 1%
glutamine, and 1% penicillin-streptomycin solution (1:1). Actinomycin
D (25 µl; 30 mg/ml) was added to each well followed by samples of
filtered rat plasma (50 µl) or purified recombinant human TNF-
as
standards (R&D Systems, Minneapolis, MN). The cells were incubated for
24 h at 37°C, and then 25 µl of sterile MTT were added to each
well. After incubation for 3 h at 37°C, the reaction was stopped by
adding 100 µl lysis buffer (20) and the plates were read at 620 nm
using a spectrophotometric plate reader. One unit of TNF-
activity
was defined as the concentration at which 50% of the cells were lysed.
Steady-state liver TNF--
and IL-10 mRNA levels
by semiquantitative RT-PCR.
Total RNA was extracted from liver tissue by the acid guanidinium
isothiocyanate method (5). The final RNA concentrations were determined
by absorbance using a GeneQuant spectrophotometer (Pharmacia,
Piscataway, NJ). The reverse transcription and PCR reactions were
performed as described previously by us (27) using the "primer
dropping" method of Wong et al. (34). Briefly, cDNA was generated by
a reverse transcription reaction by incubating 1 µg of total RNA,
1× PCR buffer (in mM, 10 Tris · HCl, pH 9.0, 50 KCl, and 1.5 MgCl), 1 nM each of deoxynucleotide triphosphates (dATP,
dGTP, dCTP, and dTTP), 20 units placental ribonuclease inhibitor (RNA guard, Pharmacia), 160 units of
Superscript-reverse transcriptase (GIBCO-BRL, Burlington, ON,
Canada), and 100 pmol of random hexamer oligodeoxynucleotides
(Pharmacia). Reaction mixtures were preincubated 10 min at 21°C
before cDNA synthesis. The reverse transcription reactions were carried
out for 50 min at 42°C and were heated to 95°C for 5 min to
terminate the reaction. Reactions were performed in an
Amplitron I thermal cycler (Barnstead/Thermolyne, Dubuque, IA).
Adrenalectomy studies. To further assess the role of endogenous glucocorticoids in CCl4-induced hepatotoxicity we used rats completely devoid of endogenous glucocorticoids due to adrenalectomy. Adrenalectomized and sham adrenalectomized rats were obtained from the supplier and were studied 3-4 days after their adrenalectomy or sham adrenalectomy. Adrenalectomized rats were maintained with 0.9% saline for drinking water. Adrenalectomized and sham adrenalectomized rats were treated with CCl4 (0.3 ml/200 g body wt by gavage) and mortality recorded over the ensuing 24 h. Another group of adrenalectomized rats was pretreated with corticosterone suspended in 500 µl of polyethylene glycol 300 (Sigma) at a dose of 10 mg/kg given subcutaneously at the time of CCl4 gavage and 12 h later. This dose of corticosterone produces stress levels of circulating corticosterone in adrenalectomized rats (12).
Liver histology. Liver tissue was collected in Formalin, sectioned, and stained with hematoxylin and eosin. Liver sections were examined under light microscopy in a blinded fashion.
Statistical analyses.
Data are expressed as means ± SE. For comparisons between two means
a Student's t-test was performed. For
comparisons among more than two means an ANOVA followed by
Student-Newman-Keuls test was performed. For comparison of mortality
data a Fishers exact test was performed. Statistical significance was
achieved if P 0.05.
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RESULTS |
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CCl4-induced corticosterone secretion.
Plasma corticosterone levels were similar in oil-gavaged control rats
12 and 24 h after gavage (Fig. 1). However,
CCl4 treatment resulted in
striking elevations in plasma corticosterone levels at all time points
after gavage compared with control levels at similar time points (Fig.
1).
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RU-486 treatment and CCl4-induced
hepatotoxicity.
Serum ALT levels were significantly elevated 24 h after
CCl4 treatment in both vehicle-
and RU-486-treated rats compared with basal levels (Fig.
2). However, serum ALT levels were
strikingly higher in RU-486-treated rats compared with vehicle-treated
rats 24 h after CCl4 gavage (Fig.
2).
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Glucocorticoid secretion and survival after
CCl4.
Adrenalectomized and sham adrenalectomized rats were gavaged with
CCl4 and subsequently assessed 12 and 24 h later for survival. Throughout the 24-h observation period no
CCl4-treated sham
adrenalectomized rats died (n = 7). However, by 24 h after CCl4
administration five of seven adrenalectomized rats had died
(P 0.02). Administration of corticosterone to adrenalectomized
CCl4-treated rats completely prevented CCl4-induced mortality
in this group (n = 7).
Hepatic TNF- mRNA expression and plasma
TNF-
levels.
The bioassay used in this study to detect circulating TNF-
is very
sensitive, and serum levels of TNF-
in control rats similar to those
detected in our experiments have been previously reported using this
assay (10). Plasma TNF-
bioactivity increased significantly and to a
similar degree 12 h after CCl4
gavage in vehicle- and RU-486-treated rats (Fig.
4). Plasma TNF-
bioactivity, however, had returned to levels similar to control rats by 24 h in both vehicle-
and RU-486-treated animals (Fig. 4).
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Hepatic IL-10 mRNA expression and plasma IL-10 levels.
IL-10 is an anti-inflammatory cytokine that has recently been shown to
be capable of protecting the liver from experimental hepatotoxicity
(17, 18). Furthermore, recent evidence suggests that glucocorticoids
can induce IL-10 synthesis and release (25, 30, 32). IL-10 was
undetectable in plasma of control rats, consistent with observations of
others (18). However, CCl4
treatment resulted in a striking increase in plasma IL-10 levels by 12 h and this remained unchanged at 24 h
post-CCl4 (Fig.
6). Rats pretreated with RU-486
demonstrated a markedly attenuated increase in plasma IL-10 levels in
response to CCl4 12 h after
CCl4 treatment (Fig. 6). By 24 h
the plasma IL-10 levels in RU-486-pretreated
CCl4-gavaged rats were similar to
levels observed in CCl4-gavaged
rats which were not pretreated with RU-486 (Fig. 6).
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DISCUSSION |
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Acute hepatocellular necrosis induced by CCl4 is a widely used model of acute liver injury (35). We have shown that acute hepatic inflammation induced by CCl4 is a potent activator of endogenous glucocorticoid secretion and that enhanced glucocorticoid release plays a significant modulatory role with respect to the development of hepatic inflammation and overall survival after CCl4 treatment in rats. Furthermore, endogenous glucocorticoid release appears to be necessary for early hepatic release of the anti-inflammatory cytokine IL-10 in response to acute CCl4 hepatotoxicity.
The release of endogenous glucocorticoids from the adrenal gland during the inflammatory response plays a central role in the control of inflammation (reviewed in Ref. 6). However, the role played by endogenous glucocorticoid secretion in modulating hepatic inflammation and the mechanisms underlying this modulation are poorly documented. In the current study we have identified that a striking increase in plasma corticosterone levels accompanies the hepatic inflammatory response due to CCl4 administration. Moreover, the rise in plasma corticosterone levels precedes histological or biochemical indexes of CCl4-induced liver damage, suggesting that endogenous glucocorticoid secretion may modulate CCl4-induced hepatotoxicity. This hypothesis was confirmed by the demonstration of enhanced CCl4-mediated lethality in adrenalectomized rats (which is completely prevented by corticosterone treatment) and an augmentation of the biochemical and histological progression of CCl4-induced hepatotoxicity in rats where endogenous glucocorticoid receptors had been blocked by RU-486 treatment (RU-486 is a glucocorticoid receptor antagonist; 15). RU-486 is also a progesterone receptor antagonist (18). Given that progesterone can modulate inflammation by repressing NF-kappa B activity (reviewed in Ref. 31), some of the biological effects observed in our studies using RU-486 may possibly be due in part to effects of RU-486 on progesterone-mediated responses.
Early studies have suggested that endogenous glucocorticoids may exert
some of their anti-inflammatory properties by inhibiting the production
of TNF-. Specifically, these studies demonstrated that
adrenalectomized mice treated with endotoxin produced higher levels of
TNF-
and were more susceptible to the lethal effects of endotoxin
(23, 36). CCl4-induced
hepatotoxicity is, at least in part, an endotoxin-mediated event (22).
Furthermore, neutralization of TNF-
in rats treated with
CCl4 has recently been shown to
attenuate CCl4-induced
hepatotoxicity (9). Therefore, our data demonstrating increased
lethality in adrenalectomized rats treated with
CCl4 compared with sham
adrenalectomized controls would be consistent with these previous
studies. However, we were unable to demonstrate enhanced hepatic
TNF-
mRNA expression or plasma TNF-
levels in
CCl4-treated rats in which
endogenous glucocorticoid receptors had been blocked with RU-486 (as
compared with CCl4-gavaged vehicle-treated controls). Our results with RU-486 are in agreement with previous studies in rats and mice that have shown that endogenous glucocorticoid blockade with RU-486 does not result in enhanced plasma
TNF-
levels in response to endotoxin treatment (11, 24). These
results suggest that TNF-
modulates
CCl4-induced hepatotoxicity by
direct or indirect effects that may be independent of specific serum or
hepatic levels of this cytokine. Recently IL-10 has been demonstrated
to be able to induce the production of soluble TNF-
receptors (16).
Because soluble TNF-
receptors inhibit the biological activity of
TNF-
, lower levels of hepatic IL-10 in RU-486-treated
CCl4-gavaged rats may lead to the
diminished local production of soluble TNF-
receptors within the
liver, which could potentially result in augmented TNF-
-mediated
hepatotoxicity in these animals.
Recent work has also suggested a possible beneficial role of TNF- in
the response of the liver to damage. TNF-
appears to play a critical
role in hepatic repair after injury. TNF-
stimulates hepatic DNA and
RNA synthesis and hepatic mitosis (3) and is of central importance in
hepatic regeneration after partial hepatectomy in rats (1). Moreover,
enhanced hepatic TNF-
levels have recently been shown to be
necessary for the induction of early-immediate genes and in liver
repair following CCl4-induced
hepatotoxicity (4). The discrepancy between adrenalectomy and RU-486
effects on TNF-
release has been examined by Pettipher et al. (24) who found that elevated plasma TNF-
levels in endotoxin-treated mice
are due to effects of released adrenal catecholamines, not glucocorticoids.
IL-10 is an anti-inflammatory cytokine, which plays a critical role in the natural defense against endotoxin-induced toxicity (2) and which has recently been shown to be capable of attenuating galactosamine-endotoxin- and concanavalin-A-induced liver injury in mice (17, 18). The mechanisms underlying the hepatoprotective effects of IL-10 are poorly understood. IL-10 may attenuate CCl4-induced hepatotoxicity through its well-documented ability to decrease the production of many proinflammatory cytokines and chemokines (reviewed in Ref. 26). Furthermore, IL-10 can inhibit the recruitment of neutrophils to sites of inflammation by downregulating the expression of endothelial cell adhesion molecules (14). Because neutrophils are known to contribute to CCl4-mediated hepatotoxicity (35), diminished neutrophil accumulation in the liver would be expected to attenuate CCl4-induced liver damage.
The liver appears to be the major source of circulating IL-10 (33). Moreover, glucocorticoids enhance IL-10 secretion from Th1 lymphocytes, and the administration of glucocorticoids to patients can increase serum IL-10 levels (25, 30, 32). Therefore, endogenous glucocorticoids may modulate inflammatory processes, at least in part, by enhancing IL-10 production. Our findings are consistent with this hypothesis. Endogenous IL-10 mRNA expression and IL-10 release were strikingly attenuated 12 h after CCl4 administration in rats receiving RU-486. By 24 h after CCl4 administration in RU-486-treated rats, plasma IL-10 levels were similar in RU-486 pretreated and nonpretreated groups, despite the observation that hepatic IL-10 mRNA levels remained low 24 h after CCl4 in RU-486-treated animals. Given that IL-10 has known hepatoprotective effects against hepatotoxicants (17, 18) these data suggest that a relative lack of glucocorticoid-driven endogenous IL-10 secretion early in the course of acute CCl4 hepatotoxicity may predispose rats to more profound hepatic damage induced by hepatotoxicants such as CCl4. The finding that at 24 h post-CCl4 treatment plasma IL-10 levels were similar in RU-486-pretreated and nonpretreated rats, despite a persistent decrease in hepatic IL-10 mRNA expression in the RU-486- pretreated rats, suggests that endogenous glucocorticoids have a complex modulatory role with respect to IL-10 production. Specifically, despite low hepatic mRNA levels in RU-486-treated rats 24 h after CCl4 gavage, IL-10 secretion appears to have normalized. However, given that serum transaminase levels and histological changes are significantly worse in CCl4-gavaged RU-486-treated rats (compared with rats not treated with RU-486) 24 h after CCl4 treatment, the early hepatic IL-10 responses may be of critical importance with respect to hepatotoxicity evident at later time points.
In summary, we have demonstrated that acute liver necrosis due to
CCl4 is associated with enhanced
endogenous glucocorticoid secretion, which attenuates the early hepatic
inflammatory response through a mechanism that appears to be
independent of hepatic and serum TNF- levels and possibly by
enhancing local IL-10 production.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stefan Urbanski for assistance with the histological assessments.
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FOOTNOTES |
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M. G. Swain is an Alberta Heritage Clinical Investigator and a Medical Research Council (MRC) Scholar and J. Wallace is an Alberta Heritage Scientist and an MRC Senior Scientist. These studies were performed with an operating grant from the MRC of Canada to M. G. Swain.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. G. Swain, Liver Unit, Gastroenterology Research Group, Univ. of Calgary, Calgary, Alberta, Canada T2N 4N1.
Received 17 March 1998; accepted in final form 1 October 1998.
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