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Regular Articles |
From the Departments of Gastroenterology*
and
Surgery,
Pancreatic Research Group, Prince of
Wales Hospital, University of New South Wales, Sydney; and the
Department of Medicine,
Hepatic Fibrosis
Group, Clinical Sciences Unit, Queensland, Institute of Medical
Research and the University of Queensland Brisbane, Australia
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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-smooth muscle actin (SMA), desmin, and
platelet-derived growth factor receptor type ß (PDGFRß). The
relationship of fibrosis to stellate cell activation was studied by
staining of serial sections for SMA, desmin,
PDGFRß, and collagen, and by dual-staining for SMA
plus either Sirius Red or in situ hybridization for
procollagen 1 (I) mRNA. The cellular source of TGFß
was examined by immunohistochemistry. The histological appearances in
the TNBS model resembled those found in human chronic pancreatitis.
Areas of pancreatic fibrosis stained positively for Sirius Red and
collagen type I. Sirius Red staining was associated with
SMA-positive cells. SMA staining colocalized with
procollagen 1 (I) mRNA expression. In the rat
model, desmin staining was associated with PDGFRß in areas of
fibrosis. TGFß was maximal in acinar cells adjacent to areas of
fibrosis and spindle cells within fibrotic bands. Pancreatic stellate
cell activation is associated with fibrosis in both human pancreas and
in an animal model. These cells appear to play an important role in
pancreatic fibrogenesis.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In contrast to the pancreas, the process of fibrogenesis has been
closely studied in the liver. Stellate cells (previously known as Ito
cells, vitamin A storing cells, or lipocytes1
) are now
known to play a major role in the production of hepatic fibrosis and
are the major source of collagen and other extracellular matrix
proteins in liver disease.2
In the normal liver, stellate
cells may be identified by the presence of fat droplets containing
vitamin A3
and by positive staining for desmin, a
cytoskeletal intermediate filament protein.4
When activated
during liver injury, stellate cells undergo both morphological and
functional changes. The cells enlarge, proliferate, and lose the
vitamin A-containing lipid droplets.3
Activated stellate
cells exhibit positive staining for the cytoskeletal protein alpha
smooth muscle actin (SMA) and become responsive to cytokines such as
platelet-derived growth factor (PDGF)3
and transforming
growth factor-ß (TGFß).5
Expression of extracellular
matrix proteins, especially collagen types I and III,2
is
increased.
Similar cells have recently been identified and isolated from the
pancreas, and have been termed pancreatic stellate cells
(PSCs).6,7
In the normal rat pancreas, stellate cells stain
positively for desmin but do not stain for SMA, indicating a
quiescent, nonactivated state.6
Recent in vitro
studies of cultured pancreatic stellate cells have demonstrated that
these cells exhibit morphological and functional features similar to
cultured hepatic stellate cells, including positive SMA staining
after a period of time in culture, increased proliferation in response
to PDGF, and increased collagen synthesis in response to
TGFß.8
The relationship of PSCs to pancreatic fibrosis in vivo remains to be established. Wells and Crawford have recently highlighted the need for morphological studies in pancreatic fibrosis to examine the role of the PSCs in fibrosis and their relationship to the profibrogenic cytokines PDGF and TGFß.9 Therefore, the aim of this study was to determine whether pancreatic stellate cell activation occurs during pancreatic fibrogenesis in vivo. This has been studied using an animal model of pancreatic fibrosis and the findings have been compared with those in human pancreatic fibrosis.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Pancreatic fibrosis was induced in male Sprague-Dawley rats by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct by a modification of the method described by Puig-Divi et al.10 Male Sprague-Dawley rats (350–500 g, n = 24) were fed standard rodent chow and tap water ad libitum until the day before surgery. Animals were fasted overnight but given free access to water. Anesthesia was induced by inhalation of 4% halothane in 100% O2 for 10 minutes and maintained with 2% halothane. During closure of the abdominal wound, halothane was ceased and O2 was given alone. Heat loss was prevented during the operation by placing the animals on a warming tray and using a hot incandescent light, and postoperatively by using an infrared warming lamp until the animals were awake. A single subcutaneous dose of ceftriaxone 10 mg (Roche Products Pty Ltd., Dee Why, NSW, Australia) was given. The abdomen was shaved, prepared with povidone-iodine, and covered with a fenestrated sterile drape. Using sterile technique, a midline upper abdominal incision was made. The entry point of the pancreatic-biliary duct into the duodenum was identified and the duodenum was opened through a horizontal 0.5-cm antimesenteric incision. The ampulla of Vater was cannulated with polyethylene tubing (O.D. 0.62 mm; Dural Plastics, Auburn, NSW, Australia) and sutured so that the tip lay 3–5 mm within the pancreatobiliary duct. The pancreato-biliary duct was occluded with a vascular clamp at the hilum of the liver to prevent entry of the infusate into the liver. 2% TNBS (Sigma Chemical Co., St Louis, MO) solution in phosphate-buffered saline (PBS, pH 8.0) with 10% ethanol was infused for 60 minutes to a total volume of 0.4 ml (Ohmeda 9000 syringe pump). Control rats (n = 4) were infused with the same volume of 10% ethanol in PBS without TNBS. The hilar clamp was removed and, after a 5-minute washout period, the cannula was removed. The duodenotomy was closed with a single interrupted layer of absorbable sutures and the abdominal wound was closed in two layers. Bupivocaine (0.25%, 1.5 ml) was infiltrated into the abdominal wound for postoperative analgesia and normal saline (30 ml/kg body weight) given by subcutaneous injection to maintain hydration.
Postoperatively, rats were transferred to individual cages and were given O2 by mask until awake and mobile. Rats were fasted for 72 hours, but allowed free access to water after 48 hours. Hydration was maintained by twice daily subcutaneous injections of sterile fluid (60 ml/kg/day; normal saline alternating with 4% dextrose containing 0.18% saline) until the animal began drinking voluntarily. Analgesia with subcutaneous buprenorphine 0.1 ml was given when necessary for postoperative analgesia. Standard rat chow and water were freely available from the third day and weight gain was recorded weekly.
Animals were killed by carbon dioxide inhalation at 4 weeks. The pancreas was removed and portions were fixed in 10% neutral buffered formalin for histology and embedded in OCT compound for frozen sections.
Human Specimens
Paraffin-embedded surgical specimens were obtained from six patients who had undergone pancreatic resection for chronic alcoholic pancreatitis.
Histological Studies
Rat Pancreas
Paraffin sections of rat pancreatic tissue were stained by
hematoxylin and eosin and for collagen using Sirius Red.11
Immunohistochemistry was performed for collagen type I, -smooth
muscle actin (SMA), and TGFß using paraffin sections, and for
desmin, glial fibrillary acidic protein (GFAP), and platelet-derived
growth factor receptor types and ß (PDGFR and PDGFRß) using
frozen sections. The relationship between pancreatic fibrosis and
pancreatic stellate cell activation was studied in rats by staining of
serial sections for SMA, desmin, and collagen and by dual-staining
of sections for SMA plus Sirius Red for collagen protein or SMA
plus in situ hybridization for procollagen 1
(I) mRNA.
Human Pancreas
Paraffin sections obtained from human alcoholic pancreatitis were
stained with hematoxylin and eosin and Sirius Red. Immunohistochemistry
was performed for SMA and for the profibrogenic cytokine
transforming growth factor ß (TGFß). Serial sections were stained
for SMA and collagen and dual staining was performed as for rat
tissues.
Immunohistochemistry
In preparation for immunostaining, paraffin sections of the pancreas were rehydrated and washed in TBS for 5 minutes three times, and frozen sections were fixed with acetone at 4°C for 10 minutes, air-dried for 10 minutes, and washed in TBS as above. Sections were incubated with 1% H2O2 for 30 minutes to block endogenous peroxidases and washed. To prevent nonspecific binding of antibody, sections were incubated for 30 minutes at room temperature with a blocking solution containing TBS, 1% bovine serum albumin (BSA), and 10% goat serum.
SMA
Paraffin sections of the pancreas were prepared for immunostaining
as described above and then incubated at room temperature for 30
minutes with the anti-SMA primary antibody (mouse monoclonal
antibody, clone 1A4; Sigma), diluted 1:100 in the blocking solution.
After further washes, the secondary antibody (horseradish
peroxidase-conjugated goat anti-mouse IgG; Sigma) was applied in a
dilution of 1:100 for 30 minutes at room temperature. After further
washes, the color was developed using the DAKO liquid with
3,3-diaminobenzidine tetrahydrochloride (DAB) substrate chromogen
system (DAKO, Botany, Australia). Sections were counterstained with
Mayer's hematoxylin (Sigma) for 5 minutes. Sections incubated with an
isotype control monoclonal IgG or without primary antibody were
included in each staining experiment as negative controls.
Desmin
Immunohistochemistry for desmin was performed using frozen
sections of the pancreas. The primary antibody used was a mouse
monoclonal antibody (clone de-u-10; Sigma) and the same types of
negative controls were used as described for SMA above. Colour was
developed using the DAKO liquid with 3,3-diaminobenzidine
tetrahydrochloride (DAB) substrate-chromogen system (DAKO,
Botany, Australia) and sections were subsequently counterstained with
Mayer's hematoxylin (Sigma).
PDGFRß
Frozen sections of the pancreas were used for PDGFRß
immunostaining. The primary anti-PDGFRß and anti-PDGFR antibodies
were rabbit polyclonal antibodies (1:50; Santa Cruz Biotechnology,
Santa Cruz, CA). The antibodies were prepared in 10% swine serum in
1% BSA and TBS and applied overnight at 4°C. The secondary antibody
was alkaline phosphatase-conjugated swine anti-rabbit IgG diluted 1:100
in the appropriate blocking solution. After TBS washes, the colour was
developed using the Sigma Fast Red naphthol substrate system according
to the manufacturer's instructions. Sections incubated with preimmune
host serum were used as negative controls.
Collagen Type I
The collagen type I antibody was a rabbit polyclonal antibody (specific for rat collagen type I, no cross-reactivity with collagen types III or IV; Amrad Pharmacia Biotech, Victoria, Australia) which was diluted 1:50 in 10% swine serum in TBS containing 1% BSA and incubated overnight at 4°C. The secondary antibody was alkaline phosphatase-conjugated swine anti-rabbit IgG and the chromogen was the Sigma Fast Red naphthol substrate system.
TGFß
Paraffin sections of the pancreas were subjected to antigen retrieval by heating in a microwave oven on high power for 8 minutes in 0.01 M citrate buffer (pH 6.0), then incubated with a mouse monoclonal anti-TGFß1,2,3 primary antibody to active TGFß (150 µg/mL; Genzyme Diagnostics, Cambridge, MA) for the cellular localization of TGFß protein as previously described.12 The DAKO StreptAB Complex/horseradish peroxidase kit was used as the detection system with DAB as the chromogenic substrate.
GFAP
GFAP staining was performed using a rabbit polyclonal anti-GFAP primary antibody as previously described.6
In Situ Hybridization for Detection of Procollagen a1 (I) mRNA
In situ hybridization for procollagen 1
(I) mRNA was performed on both rat and human pancreas sections using
previously described methods.12,13
Briefly, a 1500-bp
fragment of human procollagen 1 (I) cDNA was subcloned
into pGEM 11Z vector and then subjected to alkaline hydrolysis to
produce a 300-bp fragment for use in in situ hybridization.
Digoxigenin-labeled riboprobes, for sense (control) and antisense, were
produced by in vitro transcription with SP6 and T7
polymerases. In situ hybridization was performed on 5-µm
rat and human paraffin-embedded pancreas sections. After hybridization,
sections were washed to remove unbound probe and incubated with
alkaline phosphatase conjugated anti-digoxigenin polyclonal sera
(1:200) at room temperature for 2 hours. Unbound antibody was removed
by washes prior to color visualization with nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate in the dark at room
temperature for 16 hours. Unbound complex was removed by washing and
sections were subjected to immunohistochemistry for
SMA12
to determine whether procollagen 1
(I) mRNA colocalized with SMA-positive PSCs.
Ethics Approval
These studies were approved by the Animal Care and Ethics Committee of the University of New South Wales, Australia and the Human Research Ethics Committee of the South Eastern Sydney Area Health Service.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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TNBS-Treated Rats
All 24 rats treated with TNBS developed chronic pancreatitis with
fibrosis (Figure 1, A and B
, is
representative of the changes seen in all animals). The lesions were
focal and, as reported by Puig-Divi et al,10
the foci
varied with respect to size and number. The major histopathological
features included periductal and intralobular fibrosis associated with
a mononuclear inflammatory infiltrate and segmental glandular atrophy.
Where periductal disease extended into pancreatic lobules, fibrosis and
inflammatory cells were typically seen in the peri-acinar region
(Figure 1B)
. In severe cases, the enclosed acini were atrophic and
surrounded by fibrotic bands. Areas of fibrosis stained strongly for
the collagen stain Sirius Red (Figure 1C)
.
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The histological appearance of the pancreas from these subjects
was typical of the well characterized features of alcoholic
pancreatitis. The main characteristics were acinar atrophy associated
with intralobular, interlobular, and periductular fibrosis (Figure 2A)
. Intralobular fibrosis was most
evident in the peri-acinar regions and was associated with atrophy of
adjacent acinar tissue. These histological features, particularly the
periductular and periacinar distribution of fibrosis, resembled those
found in TNBS-induced pancreatitis in rats. As with rat pancreatic
fibrosis, areas of fibrosis in human tissue stained strongly for Sirius
Red (Figure 2B)
and also stained strongly for collagen type I by
immunohistochemistry (Figure 2C)
.
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SMA
SMA has recently been shown to be an excellent marker for the
activated PSC phenotype in vitro.6
In this
study, activated stellate cells were demonstrated both by the stellate
morphology and by the expression of SMA. This marker was strongly
positive for stellate cells in pancreas of both rats and humans
(Figures 1D and 2D)
. The peri-acinar distribution of SMA-positive
PSCs is clearly demonstrated at higher magnification (Figure 2E)
.
Desmin
This study confirmed our recent observations that desmin is a
useful marker for stellate cells of rat pancreas both in
vitro and in vivo.6
Cells that were
strongly positive for desmin were observed in fibrotic areas of rat
pancreas (Figure 4B)
. At higher magnification, the stellate morphology
of these desmin-positive cells was evident (data not shown). In
contrast to the rat pancreas, no desmin staining was observed in human
pancreas, using either formalin-fixed, paraffin-embedded tissue or
frozen sections of unfixed tissue.
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No staining was seen for GFAP in pancreatic fibrosis tissues. The methods used were validated by using a control positive tissue. Staining rat liver tissue for GFAP yielded the expected pattern of stellate cell staining.
Relationship between Activated Stellate Cells and Pancreatic Fibrosis
Dual staining techniques and staining of serial sections revealed
a strong association between activated stellate cells and fibrosis. In
the rat pancreas, Sirius Red collagen staining was closely associated
with SMA-positive cells in serial sections (Figure 1, C and D)
.
Desmin-positive cells were also closely associated with areas of
pancreatic fibrosis in serial sections (Figure 4, A and B)
. The
relationship between pancreatic fibrosis and stellate cells was most
clearly demonstrated in human pancreas by dual staining for collagen
and SMA (Figure 2E)
.
Identification of Activated PSCs as the Cellular Source of
Procollagen 1 (I) mRNA Expression
In both rat and human pancreas, blue staining for procollagen mRNA
was restricted to spindle-shaped cells in areas of fibrosis (Figure 3, A–C)
. No such staining was observed in
acinar and ductular cells. Using the in situ hybridization
technique in combination with immunostaining for SMA, a striking
colocalization of procollagen mRNA and SMA staining could be
observed in the majority of stellate cells, although some cells
appeared to stain positively for only one or other of the two markers.
There are two possible explanations for the latter observation:
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SMA is one of the earliest events in stellate cell activation,
occurring before the deposition of collagen.2
Thus at any
point in time during fibrogenesis, there will be some stellate cells in
the early phase of activation (expressing only SMA) while others
will be in the late phase of activation expressing marked levels of
both SMA and collagen mRNA.12,14,15
It is possible that
a similar dynamic process occurs during pancreatic fibrogenesis,
accounting for the lack of collagen mRNA staining in some cells.
2) The star-shaped morphology of pancreatic stellate cells (a central
cell body containing the nucleus and numerous cytoplasmic processes
extending outward from the cell body) means that in any one plane of
sectioning, there will be some cells that are represented only by their
cytoplasmic processes (where SMA staining is maximal) and others
that show only the cell body and nucleus (where staining for collagen
mRNA is maximal). This would explain why some cells appear to be
positive for collagen mRNA and not SMA and vice versa.
Relationship of Cytokine Staining to Fibrosis
PDGFRß
Positive PDGFRß staining was closely associated with areas of
fibrosis in rat pancreas as evident from serial sections (Figure 4
A
and C) but was absent in nonfibrotic
areas of the pancreas. PDGFRß staining was also closely associated
with desmin staining in fibrotic areas (Figure 4, B and C)
, suggesting
that PSCs express the PGDFRß receptor. No staining for PDGFR was
observed (data not shown) in the pancreas.
TGFß
Significant expression of TGFß was observed in fibrotic human
pancreas, particularly within acinar cells adjacent to fibrotic bands
(Figure 2F)
. Acinar cells remote from fibrosis did not stain
positively. In addition, there was positive, albeit weak, TGFß
staining in spindle-shaped cells within the bands of pancreatic
fibrosis (Figure 2F
, arrows). TGFß expression in rat pancreas was
also observed to a lesser extent.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recent studies in the liver have established that stellate cells, when
activated by profibrogenic mediators, play a key role in hepatic
fibrogenesis by synthesizing and secreting increased amounts of
extracellular matrix proteins.2,16,17
The findings of the
present study provide, for the first time, strong in vivo
evidence in support of a similar process in pancreatic fibrogenesis.
First, positive staining for the cytoskeletal protein desmin, a
stellate cell marker, was found to be concentrated within areas of
fibrosis in the pancreas, suggesting increased replication of stellate
cells during fibrogenesis. Second, dual staining techniques
demonstrated colocalization of SMA staining (indicating activated
stellate cells) and Sirius Red staining for collagen protein; this
observation provides circumstantial evidence to support the concept
that activated stellate cells may be involved in collagen production
during pancreatic fibrogenesis. Third and perhaps most important, a
combination of immunostaining for SMA and in situ hybridization for
procollagen mRNA (Figure 3, A–C)
demonstrated conclusively that it was
activated stellate cells that were the principal source of collagen
production in the fibrotic pancreas.
The advantage of the rat model of pancreatic fibrosis used in this study was that a substantial degree of pancreatic fibrosis was induced within a relatively short period of 4 weeks. Furthermore, the peri-acinar pattern of fibrosis produced in this model was very similar to that seen in human chronic pancreatitis. The presence of fibrosis in the peri-acinar region is in keeping with the concept that PSCs make an important contribution to fibrogenesis, since these cells have been shown to be situated in the peri-acinar region both in rat pancreas (by desmin staining)6 and in human pancreas (by electron microscopy).18 Although desmin is a reliable marker of quiescent stellate cells in rat pancreas, no such immunohistochemical marker has yet been identified for quiescent human pancreatic stellate cells. Therefore, the rat model provides a useful tool for future in vivo studies of the biology of stellate cells, including characterization of the process of transformation from a quiescent to an activated state, identification of factors that may activate or inactivate these cells, and delineation of the temporal relationship between stellate cell activation and production of fibrosis in the pancreas.
Immunohistochemical studies demonstrated that the fibrotic bands in both rat and human pancreatic tissue contained type I collagen. This observation may be of particular relevance to fibrogenesis, because previous studies have reported that the change in collagen synthesis from nonfibrillar collagens to fibril-forming collagens, notably type I, is an important step in hepatic fibrosis2 as well as in experimental and human pancreatic fibrosis.19,20
This study also evaluated the role of two profibrogenic cytokines in
pancreatic fibrosis. PDGF is a dimer of two types of peptide chains,
named A and B peptides, resulting in the isoforms PDGF AA, PDGF BB, and
PDGF AB.21
The PDGF receptor is also composed of two types
of peptides ( and ß) assembled into a dimer (, ß, or
ßß). The receptor recognizes both A and B chains of PDGF,
whereas the ß receptor recognizes only the B chain of
PDGF.21
PDGF BB is known to stimulate hepatic stellate
cells to proliferate, resulting in increased responsiveness to other
proinflammatory cytokines.22
The proliferative effect of
PDGF BB has also recently been demonstrated in an in vitro
study using cultured pancreatic stellate cells.8
The
present study has demonstrated that the expression of PDGFRß, but not
PDGFR, is closely associated with areas of pancreatic fibrosis. This
finding is consistent with reports demonstrating increased expression
of PDGF ligand type BB and receptor type ßß in chronic
pancreatitis23
and selective up-regulation of the type ß
receptor in activated hepatic stellate cells.24
The other profibrogenic cytokine examined in this study was TGFß. A number of studies have previously demonstrated increased expression of TGFß in human chronic pancreatitis.25,26 van Laethem et al have reported positive TGFß staining of acinar cells and spindle-shaped cells within fibrotic bands.25 In contrast, Slater reported maximal TGFß staining in ductal and ductular epithelial cells with no staining in spindle-shaped cells.26 Up-regulation of TGFß mRNA levels has also been reported in caerulein-induced pancreatitis in rats.27-29 The importance of TGFß in pancreatic fibrosis is shown by studies in which collagen synthesis in a rat model of recurrent pancreatitis was stimulated by exogenously administered TGFß30 and inhibited by administration of a TGFß-neutralizing antibody.31 Our laboratory has recently reported that cultured PSCs respond to exogenous TGFß with increased collagen synthesis.8 In the present study, increased TGFß expression was demonstrated within acinar cells adjacent to areas of fibrosis. This observation suggests that acinar cells may be a significant cellular source of TGFß in pancreatic fibrogenesis. We propose that TGFß is an important profibrogenic cytokine in vivo that appears to act by stimulating collagen production by pancreatic stellate cells close to TGFß-positive acinar cells.
In conclusion, this study has provided significant in vivo
evidence in support of a major role for pancreatic stellate cells in
pancreatic fibrogenesis. PSCs were found to be the principal cellular
source of type I collagen in pancreatic fibrosis in both humans and in
an experimental animal model. A potential mechanism for the observed
stellate cell activation may involve TGFß released from injured
acinar cells leading to increased expression of procollagen
1 (I) mRNA in PSCs, and ultimately increased collagen
protein synthesis by these cells.
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Acknowledgements |
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Footnotes |
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Supported by research grants from the Gastroenterological Society of Australia, the National Health and Medical Research Council of Australia, the Department of Veterans' Affairs, and the Clive and Vera Ramaciotti Foundation.
Accepted for publication June 18, 1999.
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