1 Department of Gastroenterology, 2 Hôpital Erasme and Laboratory of Experimental Gastroenterology, and 3 Laboratory of Experimental Cytology and Cancerology, Université Libre de Bruxelles, B 1070 Brussels, Belgium; and 4 Department of Medical Cell Biology, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom
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ABSTRACT |
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Interleukin (IL)-10, a potent
anti-inflammatory cytokine, limits the severity of acute
pancreatitis and downregulates transforming growth factor (TGF)-
release by inflammatory cells on stimulation. Proinflammatory
mediators, reactive oxygen species, and TGF-
can activate pancreatic
stellate cells and their synthesis of collagen I and III. This study
evaluates the role of endogenous IL-10 in the modulation of the
regeneration phase following acute pancreatitis and in the development
of pancreatic fibrosis. IL-10 knockout (KO) mice and their C57BL/6
controls were submitted to repeated courses (3/wk, during 6 wk,
followed by 1 wk of recovery) of cerulein-induced acute pancreatitis.
TGF-
1 release was measured on plasma, and its pancreatic
expression was assessed by quantitative RT-PCR and
immunohistochemistry. Intrapancreatic IL-10 gene expression was
assessed by semiquantitative RT-PCR, and intrapancreatic collagen content was assessed by picrosirius staining. Activated stellate cells
were detected by immunohistochemistry. S phase intrapancreatic cells
were marked using tritiated thymidine labeling. After repeated acute
pancreatitis, IL-10 KO mice had more severe histological lesions and
fibrosis (intrapancreatic collagen content) than controls. TGF-
1 plasma levels, intrapancreatic transcription, and
expression by ductal and interstitial cells, as well as the number of
activated stellate cells, were significantly higher. IL-10 KO mice
disclosed significantly fewer acinar cells in S phase, whereas the
opposite was observed for pseudotubular cells. Endogenous IL-10
controls the regeneration phase and limits the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice.
experimental chronic pancreatitis; pancreatic fibrosis; transforming growth factor-; pancreatic stellate cells
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INTRODUCTION |
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VARIOUS OVERLAPPING
MECHANISMS have been shown to be involved in the pancreatic
fibrogenesis. They included the necrosis-fibrosis sequence
(19), a direct inflammatory pathway (4, 8,
38), the ischemia-reperfusion phenomenon
(30), alcohol toxicity (directly and/or via reactive
oxygen intermediates and acetaldehyde) (1, 25), and T
lymphocyte-induced cytotoxicity (7, 16, 37). The
pancreatic stellate cells (PSC), similar to hepatic stellate cells,
also play a major role in this process (6). These
intrapancreatic resident periacinar and interlobular fibroblastlike
cells disclose the characteristics of myofibroblasts (2,
6). On stimulation, they are able to express -smooth muscle
actin (
-SMA), to have contractile properties, to increase their
synthesis of type I (and also types III and IV) collagen, laminin, and
fibronectin, to form microfilaments, and to increase their own cytokine
synthesis leading to autocrine regulation (2, 6). So far,
identified major activators of PSC are transforming growth factors
(TGF)-
and -
1, platelet-derived growth factor,
reactive oxygen intermediates, ethanol, and acetaldehyde (3, 4,
6). Activated PSC have been identified in human and experimental
fibrotic areas from chronic pancreatitis tissues (15, 17)
and seem to play a major role in the development of fibrosis in chronic
pancreatitis (2-4, 6).
On the other hand, TGF-1, a direct PSC activator, is
expressed by ductal and interstitial cells in chronic pancreatitis
(20, 38) and seems to be involved in glandular atrophy
through an antiproliferative effect on acinar cells (3, 6)
observed in chronic pancreatitis. Interestingly, TGF-
1
is also released during the early course of acute pancreatitis and
during its regeneration phase, playing a major role in the regulation
of repair mechanisms (12, 14, 32).
Interleukin (IL)-10 is a potent anti-inflammatory cytokine (27) released during the course of experimental acute pancreatitis, which is known to limit its severity by downregulating proinflammatory mediator release (21, 33, 39, 40). IL-10 also has direct antiproliferative and antifibrotic properties (24, 27, 31, 36).
The present study aims to assess the potential role of endogenous IL-10 in modulating the pancreatic repair phase following acute pancreatitis and the development of chronic pancreatic lesions and fibrosis in an experimental murine model of chronic lesions induced by repeated acute pancreatitis.
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METHODS |
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Animals and Experimental Design
Eight- to ten-week-old C57BL/6 (IFFA-CREDO, Brussels, Belgium) and IL-10 knockout (KO)-targeted mutant (IL-10 KOAcute pancreatitis was induced by five intraperitoneal injections of 50 µg/kg cerulein (Pharmacia-Upjohn, Brussels, Belgium) at hourly intervals. Its severity was assessed 6 h (T6) after the beginning of cerulein challenge.
For studying the regeneration phase after acute pancreatitis, mice were killed by cervical dislocation before [day 0 (D0)] and at days 1 (D1), 2 (D2), 7 (D7), and 10 (D10) after a single cerulein-induced acute pancreatitis.
Chronic lesions were induced by repeated acute pancreatitis episodes as previously described (28). Mice were submitted to three episodes of acute pancreatitis per week, during six consecutive weeks, and then killed by cervical dislocation 1 wk later.
Blood samples were obtained by direct intracardiac puncture. Pancreata
were immediately removed, embedded in Optimum Cutting Temperature
compound (Tissue-Tek, Sakura, Zoeterwoude, the Netherlands), then
frozen in liquid nitrogen and stored at 80°C until assayed (collagen measurements). Pancreatic tissues were also fixed in formaldehyde for histology, autoradiography after thymidine labeling and immunohistochemistry, or directly harvested for RT-PCR assays.
Blood Assays
Serum amylases and lipases were measured by using automated chromogenic and turbidimetric assays at 37°C. Results were expressed in international units per liter. Active TGF-Histological Grading of Pancreatic Lesions
Hematoxylin-eosin (H&E) staining was performed on 6-µm pancreatic sections. Severity of acute pancreatitis was blindly graded at T6, by a semiquantitative assessment of edema, inflammatory cell infiltrate, and acinar necrosis according to the score previously described (40).Severity of the chronic lesions was graded by a semiquantitative scoring system. Within pancreatic sections, areas of abnormal pancreatic tissue architecture were observed. These areas were more or less numerous, and this first parameter was graded as follows: 0 = absent, 1 = rare, 2 = minimal (<10%), 3 = moderate (<50%), and 4 = major (>50%) of the total parenchyma affected. Within these areas, glandular atrophy (0 = absent, 1 = minimal, 2 = moderate, and 3 = severe), presence of pseudotubular complexes (0 = absent, 1 = minimal, 2 = moderate, and 3 = major) and fibrosis (0 = absent, 2 = only within areas, and 4 = diffuse) were observed and graded as described. A total score was calculated for each pancreatic section.
Immunohistochemical Staining of Pancreatic TGF-1
and Stellate Cells
Tritiated Thymidine Pulse Labeling
To identify intrapancreatic S phase cells, mice were injected intraperitoneally with 1 µCi/g body wt (diluted in 100 µl saline total volume) [methyl-3H]thymidine (Amersham Life Science, Little Chalfont, UK) 1 h before being killed (13). Pancreas samples were fixed in formalin during 6 h. Exposure time for autoradiography was 21 days. Sections were then counterstained with H&E and mounted using Depex mounting medium (DAKO, Merelbeke, Belgium) (13). Positive nuclei (showing silver grains) were counted in 10 consecutive (nonoverlapping) high-power fields (HPF) of 2,500 µm2 each, on the same section, at a ×400 magnification. Results were expressed as number of positive cells (acinar or pseudotubular or both) per 10 HPF.TGF-1 and IL-10 Gene Expression in Pancreata
For TGF-1, quantitative RT-PCR was then performed using
the Quantitative Competitive PCR Protocol (mouse TGF-
, Maxim
Biotech, San Fransisco, CA). Results were expressed in attomoles of
TGF-
reported to
-actin for each sample.
To quantify semiquantitatively IL-10 mRNA, the ratio of densitometric
calculations of -actin to IL-10 PCR bands was calculated.
Intrapancreatic Collagen Quantification
Sirius red staining of collagen (18, 24) was used for quantification of total intrapancreatic collagen content.From each pancreas, three cryostat sections of 10 µm were cut and air dried for 20 min. After the slices were rinsed three times for 3 min in PBS, they were fixed in Susa solution (4.5 g HgCl, 0.5 g NaCl, 10 ml 20% trichloroacetic acid, 4 ml acetic acid, 20 ml formalin, and 70 ml distilled water) (5) for 1 h at room temperature and then rinsed three times for 1 min in distillated water. They were then stained in 0.1% Sirius red F3BA (chroma) in saturated picric acid solution for 45 min at room temperature. Slices were rinsed once for 40 s with water and then for 40 s in a 0.01 M HCl solution. After 2 h air drying, slices were mounted using Depex mounting medium (DAKO).
Quantitative analysis of collagen was performed by morphometric analysis (11, 18). A digitized picture of each pancreatic section, viewed through a Zeiss Axioplan microscope (Carl Zeiss, Iena, Germany) equipped with a ×4 objective lens, was transmitted by a Sony 3-CCD video camera to a Dell 300-MHz PC equipped with a KS 300 imaging system (Kontron Elektronic, Munich, Germany). The total amount of collagen stained on each submitted section was calculated by the computer via the digitalized image as follows. In the first step, pancreas was distinguished from the background according to a difference in light density, and a measurement of the total pancreatic tissue area was performed. In the second step, the amount of collagen (stained in red) was also measured and was finally expressed as a percentage of the total pancreatic surface.
Statistical Analysis
Data are expressed as means ± SE. Each experiment relies on 15 mice per group and per time point. Statistical analysis was performed using Wilcoxon's, ANOVA, and Neuman-Keuls correction for multiple comparisons, when appropriate. P values <0.05 were considered as significant. ![]() |
RESULTS |
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Modulation of the Repair Phase After a Single Course of Acute Pancreatitis
To investigate wether a modulation of regeneration and repair phase was already present after a single course of acute pancreatitis, mice were killed before (D0) and 6 h and 1, 2, 7, and 10 days after cerulein-induced acute pancreatitis.As previously shown, IL-10 KO mice disclosed more severe acute pancreatitis 6 h after starting cerulein injections [severity score of histological lesions calculated as previously described (40); 6.3 ± 0.2 vs. 4.9 ± 0.3 for IL-10 KO and control mice, respectively; P < 0.05] and serum hydrolase levels [amylases 27302.8 ± 1008.6 vs. 17110.7 ± 1008.0 IU/ml (P < 0.05) and lipases 3657.2 ± 200.1 vs. 2130.2 ± 145.6 IU/ml (P < 0.05) for IL-10 KO and control mice, respectively].
TGF-1 Expression During Regeneration Phase
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Seven days after acute pancreatitis, TGF-1 plasma levels
were significantly higher in IL-10 KO mice (108.3 ± 5.2 vs.
83.6 ± 5.3 ng/ml for IL-10 KO and control mice, respectively;
P < 0.05; Fig. 1). Similarly, immunohistochemical
expression of TGF-
1 in both acinar and ductal cells was
more prominent (scores were 6.2 ± 0.2 and 4.2 ± 0.2 in
IL-10 KO and control mice, respectively; P < 0.05).
Return to baseline values was observed for both groups of mice on D10.
Intrapancreatic Collagen Deposition During the Regeneration Phase
Total amount of pancreatic collagen was similar in the two groups at the basal state (4.2 ± 0.3 vs. 3.9 ± 0.4% in IL-10 KO and control mice, respectively). An increase in collagen content (compared with basal values) was observed at D7 and was significantly higher in IL-10 KO (11.8 ± 0.6 vs. 9.1 ± 0.3% in IL-10 KO and control mice, respectively; P < 0.05; Fig. 2). Return to baseline values was observed for both groups of mice on D10.
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Acinar Cell Proliferation During the Regeneration Phase
At the basal state, the number of [3H]thymidine-labeled acinar cells in the S phase of mitosis was comparable in IL-10 KO and control mice (3.2 ± 0.6/10 HPF vs. 3.0 ± 0.4/10 HPF, respectively). At D7, a significant increase in this number was observed in controls, whereas it was dramatically lower in IL-10 KO mice: 12 ± 0.9/10 HPF vs. 4.0 ± 0.3/10 HPF for controls and IL-10 KO, respectively (P < 0.05; Fig. 3). Return to baseline values was observed for both groups of mice on D10.
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Endogenous IL-10 Limits Fibrosis and Modulates Pancreatic Cell Proliferation After Repeated Courses of Acute Pancreatitis
IL-10 KO mice disclosed more severe pancreatic histological
lesions, collagen deposition, and TGF-1 expression than
controls.
No death was observed in both groups of mice. After 6 wk of repeated
courses of acute pancreatitis, histological lesions (atrophy, focal
lesions, and pseudotubular complexes) and fibrosis were observed in
both groups of mice but were significantly more severe in IL-10 KO mice
[with regard to the histological score: 10.9 ± 0.7 vs. 7.9 ± 1.2 (P < 0.05; Fig.
4, A and B); with
regard to the total amount of pancreatic collagen content: 19.7 ± 1.3 vs. 15.0 ± 0.6% (P < 0.05; Fig. 4,
C and D) for IL-10 KO vs. controls, respectively].
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Endogenous IL-10 intrapancreatic gene transcription is upregulated in control mice after repeated acute pancreatitis. IL-10 gene transcription was clearly increased after repeated courses of acute pancreatitis, compared with basal state in wild-type mice [0.052 ± 0.002 vs. 0.342 ± 0.018 (P < 0.05), respectively, at baseline and after repeated acute pancreatitis].
Endogenous IL-10 controls the activation of stellate cells.
In both groups, a positive -SMA staining was observed, demonstrating
the presence of activated stellate cells (previously identified by
double-staining desmin and GFAP on serial sections).
SMA
staining was stronger for IL-10 KO mice pancreata, in which it was
predominantly located around acini and pseudotubular complexes and in
focal areas of chronic lesions. Grading scores were 4.1 ± 0.6 for
IL-10 KO mice vs. 2.4 ± 0.2 for controls (P < 0.05; Fig. 4, G and H).
Endogenous IL-10 downregulates the proliferation of acinar cells
and upregulates that of pseudotubular cells.
After 6 wk of repeated acute pancreatitis courses, both groups of mice
disclosed higher numbers of S phase-labeled acinar cells compared with
basal state (3- and 5-fold for IL-10 KO and controls, respectively).
[3H]thymidine-labeling index was significantly higher for
controls when measuring the total number of intrapancreatic-labeled
cells (acinar + pseudotubular cells: 20.8 ± 0.5/10 HPF vs.
16 ± 1.5/10 HPF; P < 0.05) and the number of
acinar-labeled cells [18.1 ± 1.6/10 HPF vs. 10.1 ± 1.1/10HPF; P < 0.05; Fig.
6, A and B] in control vs. IL-10 KO mice, respectively.
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DISCUSSION |
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The major message to be taken from this study is that, after
repeated acute pancreatitis in mice, endogenous IL-10 limits fibrogenesis, collagen deposition, and TGF-1 expression.
This is associated with a modulation of the regeneration phase
consisting of a dramatic decrease of acinar cell proliferation of IL-10
KO. IL-10 KO mice also disclose more activated stellate cells in the pancreatic parenchyma.
It was known from previous studies that both the endogenous production
of IL-10 as well as its exogenous administration downregulate the
severity of experimental acute pancreatitis. They limit acinar necrosis, parenchymal infiltration by polymorphonuclear cells and
macrophages, and release of proinflammatory mediators [including IL-1,
IL-6, tumor necrosis factor- (TNF-
) and chemokines such as
MCP-1] (21, 33, 39, 40).
After the acute pancreatitis injury, a regeneration or repair phase of
the pancreas is characterized by the decreases of inflammatory cell
infiltrate and of the release of proinflammatory mediators (9,
10, 43). A proliferation of acinar cells occurs as soon as
72 h after a cerulein-induced acute pancreatitis in rats and
persists for at least 1 wk, with restoration of the acinar cell ratio
(10, 43). TGF-1 production and release by
acinar and inflammatory cells starts 24 h after the induction of
acute pancreatitis, peaks after 48 h, and returns to basal values
only after 1-2 wk (14). Therefore,
TGF-
1 probably plays a major role in the synthesis of
collagen, which occurs during the repair phase of the pancreas, with
peaking pancreatic values at 1 wk. After a single episode of acute
pancreatitis, this collagen is destroyed and the pancreatic content
returns to preinjury values within 2 wk (10, 43). PSC are
key cells responsible for the production of collagen (essentially types
I and III) in the pancreas, and their activity is modulated in vitro by
oxidative stress, TGF-
, and TGF-
(3, 4, 6). In the
model of secretagogue acute pancreatitis (cerulein induced), the
regeneration phase leads to a complete restitution of the parenchyma
within 2 wk (even in IL-10 KO mice). The fact that acute pancreatitis
episodes were induced three times per week (i.e., mice were
rechallenged with cerulein before recovery from the previous episode)
led to an accumulation of lesions in a parenchyma still in regeneration and finally to chronic lesions in the model of chronic pancreatitis we used.
Our in vivo experiments have demonstrated that during that frame of
events, endogenous IL-10 downregulates the release and the
intrapancreatic expression of TGF-1 by acinar and
interstitial cells. This probably results from the higher severity of
the primary pancreatic injury in the absence of endogenous IL-10,
leading to a more pronounced stimulation of TGF-
1
release by proinflammatory mediators. Moreover, a direct role of IL-10
can be involved, as this cytokine has a direct inhibitory effect on
TGF-
1 production (27).
TGF-1 is the most potent activator for collagen
synthesis by PSC. Other cytokines, such as IL-6, IL-1, or TNF-
, are
known to stimulate the growth of hepatic stellate cells. However, their potential effect on PSC has not yet been demonstrated (3,
6). Because TGF-
1 production is upregulated
during the repair phase after acute pancreatitis in IL-10 KO mice, it
is not surprising to note an increase in pancreatic collagen content of
IL-10-deficient mice. However, plasma levels as well as interstitial
cell expression of TGF-
1 are higher during the
regeneration phase than during the chronic phase. This would suggest
that TGF-
1 is not the only mediator implicated in the
development of fibrogenesis observed in this model. Therefore, IL-10
can be indirectly implicated in limiting pancreatic collagen deposition
following acute pancreatitis. Moreover, it was previously shown that
IL-10 also has antifibrotic properties on fibroblastic cells by
directly downregulating procollagen I and enhancing collagenase gene
expression in vitro. It could, thereby, play in addition a direct role
in downregulating pancreatic collagen synthesis besides its
modulation of TGF-
1 secretion (9, 10, 12,
43).
After cerulein-induced acute pancreatitis, a peak of acinar cell
proliferation occurs (9), followed by the restoration of
the amount of acinar cells within 1 wk. We presently observed that 7 days after acute pancreatitis induction, the number of acinar cells in
S phase was lower in the IL-10-deficient mice despite the increased
severity of pancreatitis. After repeated courses of acute pancreatitis,
the number of acinar cells in S phase was still higher than at the
basal state but statistically lower in IL-10 KO mice. This could be
attributed to a different expression of TGF-1 in these
mice. Indeed, it is well established that TGF-
1 has a
negative effect on epithelial and acinar cell proliferation (8,
23, 35, 41). This differential effect of IL-10 could participate
in the histological remodeling occuring in chronic pancreatitis
directly or via the modulation of TGF-
1. In this
setting, our results suggest that the inbalance between TGF-
1 and IL-10 is of major importance not only for the
transformation of the interstitial cell compartment, but also for
the modulation of exocrine cells. Similarly, a loss of acinar cells and
the development of tubular structures and fibrosis were also observed
in TGF-
and -
1 transgenic mice (22, 34,
42).
Endogenous IL-10 limits the development of glandular atrophy, fibrosis,
and pseudotubular complex formation in the present model. We
hypothesized that repeated pancreatic infiltration by inflammatory
cells and in situ release of proinflammatory cytokines would lead to a
persistent activation of PSC. This could be amplified if the
TGF-1 and proinflammatory effects are not
counterbalanced by the downregulating action of endogenous IL-10.
Moreover, TGF-
1 can be upregulated via an autocrine loop
(26), and subsequently self-regulated events, such as
inflammation, tissue repair, and extracellular matrix deposition, but
also acinar cell proliferation, could evolve to uncontrolled and
persistent fibrogenesis and acinar cell atrophy in case of unbalanced
regulation and overactivation of PSC, as presently observed in IL-10 KO
mice after repeated acute pancreatitis. Mechanisms controlling this
sequence are not completely understood, especially regarding the
inbalanced regulation of inflammation by TGF-
1 and
IL-10. Moreover, through its known direct and indirect effects, IL-10
is probably one major anti-inflammatory and antifibrotic mediator
acting on PSC in the setting of a necroinflammatory pathway.
The relevance of this model of repetive acute pancreatitis episodes to human fibrogenesis is unknown. Although the pathophysiology of chronic pancreatitis is surely multifactorial, the necrosis-fibrosis sequence remains one of the most relevant theories, and it is the reason why we chose this model to investigate the possible role of endogenous IL-10 in controlling regeneration and fibrosis. We clearly observed that in the absence of IL-10, acute and chronic regulation of the fibrogenetic process are amplified and may lead to alterations consistent with those of chronic pancreatitis. However, despite the incomplete relevance of the model to human chronic pancreatitis, exogenous administration of IL-10 thus appears as a good candidate able to control excessive fibrogenesis and might be considered in the future for studiing its antifibrotic effect in human pancreatic diseases.
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ACKNOWLEDGEMENTS |
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We especially thank P. Galand and I. Salmon for helpful discussion and C. Duponchelle and F.-X. DeMoor for skillful technical assistance.
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FOOTNOTES |
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This study was supported, in part, by grants from Biotherabel and AstraZeneca Belgium and from the Belgian "Fonds National de la Recherche Scientifique."
A. Demols is a research fellow of the Fondation Erasme.
Address for reprint requests and other correspondence: A. Demols, Dept. of Gastroenterology, ULB, Hôpital Erasme, Route de Lennik, 808, B 1070 Brussels, Belgium (E-mail: ademols{at}ulb.ac.be).
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.
10.1152/ajpgi.00431.2001
Received 5 October 2001; accepted in final form 14 January 2002.
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