Departments of 1 Surgery and 2 Immunology, University of Liverpool, Royal Liverpool University Hospital, Liverpool L69 3GA, United Kingdom
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ABSTRACT |
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Inflammatory mediators including
chemokines play a critical role in acute pancreatitis. The
precise nature of early inflammatory signals within the pancreas
remains, however, unclear. We examined the ability of isolated
pancreatic acini to synthesize CC chemokine monocyte chemotactic
protein-1 (MCP-1) and CXC chemokine cytokine-induced neutrophil
chemoattractant (CINC) and the response to the secretagogue cerulein at
physiological and supraphysiological concentrations. Isolated rat
pancreatic acini maintained in short-term (48 h) primary culture
constitutively synthesized MCP-1 and CINC. Cerulein (10
7
M; supramaximal dose) increased production of MCP-1 but not CINC. Cerulein-induced increase in MCP-1 synthesis was accompanied by increase in nuclear factor (NF)-
B activation shown by EMSA.
Pretreatment with NF-
B inhibitors N-acetylcysteine (NAC)
and N-tosylphenyalanine chloromethyl ketone (TPCK) blocked
cerulein-induced NF-
B activation and abolished cerulein's effect on
MCP-1 synthesis. Pretreatment with calcium antagonist BAPTA-AM also
blocked cerulein's effect on MCP-1 synthesis. These results indicate
that isolated acini synthesize MCP-1 and CINC and support the idea of
acinar-derived chemokines as early mediators of inflammatory response
in acute pancreatitis. Although cerulein hyperstimulation increased
MCP-1 synthesis by a calcium-dependent mechanism involving NF-
B
activation, CINC synthesis was not affected. This suggests that
regulation of CC and CXC chemokines within acinar cells may be quite different.
monocyte chemotactic protein-1; cytokine-induced neutrophil
chemoattractant; nuclear factor-B.
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INTRODUCTION |
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ACTIVATION OF DIGESTIVE ENZYMES within pancreatic acinar cells is a critical initiating event in acute pancreatitis resulting in acinar cell damage and a localized inflammatory response (16, 28, 34). Secreted bioactive molecules from infiltrating leukocytes contribute to local damage and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death (5). The initial signals that recruit leukocytes into the pancreas are incompletely defined, although several inflammatory mediators have been implicated (5, 15).
The chemokines are a family of small (8-10 kDa) cytokines with activating and chemotactic effects on leukocyte subsets. CC chemokines, such as monocyte chemotactic protein-1 (MCP-1), principally affect monocytes, whereas CXC chemokines that possess the ELR motif at the amino terminal tend to act on neutrophils (1). Interleukin (IL)-8 is the best characterized of the ELR-positive CXC chemokines. Its plasma levels are elevated early in the course of acute pancreatitis and correlate with disease severity (3, 17). Treatment with a monoclonal anti-human IL-8 antibody (WS-4) was shown recently to decrease acute lung injury in experimental pancreatitis in rabbits (26). The involvement of CC chemokines in acute pancreatitis is suggested by decreased pulmonary damage in CCR1 receptor knockout mice after induction of acute pancreatitis (12).
There are important differences in the profile of chemokines seen in
different species. In the rat, for example, although there is a direct
homolog of MCP-1, there is none for IL-8. Rather, cytokine-induced
neutrophil chemoattractant (CINC), which is a homolog of the human
ELR-positive CXC chemokine growth-related oncogene- (GRO-
),
is the best characterized of the rat CXC chemokines. We recently
demonstrated (8) increased expression of MCP-1 and, to a
lesser extent, of CINC on pancreatic acinar cells by immunohistochemistry soon after induction of acute pancreatitis by
cerulein hyperstimulation or by infusion of sodium taurocholate into
the biliopancreatic duct in the rat. mRNA for CINC and MCP 1 was
detectable within pancreas as early as 1 h after induction of
pancreatitis, suggesting that both are very early mediators of the
inflammatory response in acute pancreatitis. In the present study we
show constitutive expression of MCP-1 and CINC by isolated rat
pancreatic acinar cells. In addition, we demonstrate increased production of MCP-1, but not CINC, by a nuclear factor (NF)-
B- and
Ca2+-dependent mechanism after treatment with supramaximal
doses of cerulein.
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MATERIALS AND METHODS |
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Preparation of pancreatic acini. Pancreatic acini were obtained from rat pancreas by collagenase treatment as described previously (4, 11). Briefly, pancreata from Wistar rats (100-125 g) were removed under aseptic conditions, infused with collegenase buffer A (in mM: 140 NaCl, 4.7 KCl, 1.13 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.2) containing 200 IU/ml collagenase and 0.5 mg/ml soybean trypsin inhibitor, and incubated in a shaking water bath for 10 min at 37°C. The digested tissue was passed through 50 mg/ml BSA and washed twice with collegenase buffer A before culture.
Short-term primary culture of pancreatic acini.
Pancreatic acini were suspended in Weymouth's MB 752/1 medium
containing 0.1% BSA, 0.5 mg/ml soybean trypsin inhibitor, 25 ng/ml
epidermal growth factor, and antibiotics (9). Equal
volumes (~4 × 105 cells/ml) were then aliquoted
into 24-well plates and incubated at 37°C in a humidified atmosphere
of 10% CO2-90% air. Acini were cultured for periods of
48 h. Cell viability was assessed by trypan blue exclusion.
Transmission electron microscopy. Small pieces of tissue (~1 mm) were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 1 h (minimum) at room temperature. The specimens were washed in 0.1 M cacodylate buffer (pH 7.4) for three washes of 5 min each. Specimens were then fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature and washed in 0.1 M cacodylate buffer (pH 7.4) for three washes of 5 min each. The samples were then dehydrated and embedded in epon-araldite resin. The blocks were sectioned on a Reichert Ultracut E ultramicrotome set to give 120-nm-thick sections. The sections were picked up onto 200-mesh hexagonal thin bar copper grids and stained in 2% uranyl acetate for 20 min followed by Reynolds lead citrate for 5 min. The sections were examined in a Philips CM10 transmission electron microscope operated at 80 kV. Negatives were recorded on Kodak 4489 film.
Amylase assay. Amylase activity was measured with a kinetic spectrophotometric assay. Media were incubated with the substrate 4,6-ethylidene-(G7)-p-nitrophenyl(G1)-1-D-maltoheptoside (Sigma) for 2 min at 37°C, and absorbance at 405 nm was measured every minute for a subsequent 2 min (4, 27).
MCP-1 and CINC assays. Conditioned media were assayed for MCP-1 and CINC with a sandwich ELISA according to the manufacturer's instructions. Matched antibody pairs against rat MCP-1 and recombinant rat MCP-1 were obtained from Pharmingen. Matched antibody pairs against rat CINC and recombinant CINC were obtained from R&D Systems. Briefly, anti-CINC or anti-MCP-1 primary antibody was aliquoted onto ELISA plates and incubated at 4°C overnight. Samples and standards were incubated for 2 h, the plates were washed, and a biotinylated anti-CINC or anti-MCP-1 antibody was added for 1 h. Plates were washed again, and streptavidin bound to horseradish peroxidase was added for 30 min. After a further wash, tetramethylbenzidine was added for color development and the reaction was terminated with 0.18 M H2SO4. Absorbance was measured at 450 nm.
Nuclear protein extraction.
Nuclear proteins were extracted as previously described
(23). Briefly, pancreatic acini were washed once with PBS
and the pellet was resuspended in buffer A (in mM: 50 NaCl,
10 HEPES, pH 8.0, 1 EDTA, 0.5 spermidine, and 0.15 spermine with 0.5 M
sucrose and 0.2% Triton X-100). After a 10-min incubation at 4°C,
nuclei were pelleted by centrifugation (3,500 g for 3 min at
4°C), washed once with buffer B (in mM: 50 NaCl, 10 HEPES,
pH 8.0, 1 EDTA, 0.5 spermidine, and 0.15 spermine with 25% glycerol)
and incubated for 30 min in buffer C (in mM: 350 NaCl, 10 HEPES, pH 8.0, 1 EDTA, 0.5 spermidine, and 0.15 spermine with 25%
glycerol). After a 1-min centrifugation (3,500 g, 4°C) the
supernatant was aliquoted, snap-frozen in liquid nitrogen, and stored
at 80°C.
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assays (EMSAs) were carried out
essentially as described previously (23). Reaction
mixtures (in a final volume of 10 µl) containing 4% Ficoll, 20 mM
HEPES, pH 7.5, 35 mM NaCl, 60 mM KCl, 0.01% NP-40, 2 mM
dithiothreitol, 0.1 mg/ml BSA, 1 µg Poly(dI-dC), and the indicated
amount of nuclear protein extract were incubated for 10 min at room
temperature. For supershift assays, antibodies against p50, p65, and
c-rel (Santa Cruz Biotechnology), when used, were added at
this time and the incubation period was extended to 20 min.
32P-labeled oligonucleotide (0.02 pmol) was added, and the
reaction continued for a further 20 min. In competition experiments,
unlabeled oligonucleotide was added before addition of
32P-labeled oligonucleotide. The reaction mixtures were
loaded onto 5% polyacrylamide gels containing 0.5× TBE (in mM: 89 Tris, 89 boric acid, and 0.2 EDTA). Electrophoresis was performed at
200 V in the same buffer (0.5× TBE), and the gels were then
transferred to Whatman 3MM filter paper, dried, and autoradiographed.
The following oligonucleotides were used: 5'-AGTTGAGGGGACTTTCCCAGGC-3' (containing the NF-B consensus binding site) or
5'-AGTTGAGGCCACTAACCCAGGC-3' (containing a mutated NF-
B site).
Statistics.
Results are presented as means ± SE of a typical experiment with
six replicates for each condition. Each experiment was repeated at
least three times. In Figs. 1-4 and 6-10, error bars denote
SE and the absence of such bars indicates that the SE is too small to illustrate. Initial statistical comparisons were made with a
Kruskal-Wallis test and subsequent paired comparisons with a
Mann-Whitney U-test; the level of significance was set at
5%.
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RESULTS |
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Viability and biological activity of rat pancreatic acinar cells in
primary culture.
The morphology of rat pancreatic acini in primary culture, as evidenced
by the ultramicroscopic examination of epon-embedded sections, was
unchanged up to 48 h, the latest time point studied (Fig.
1A). At 48 h >95% of
cells were viable, as determined by trypan blue exclusion. Moreover,
amylase levels as a function of DNA remained constant for up to 48 h (Fig. 1B). Amylase secretion in response to cerulein
treatment demonstrated a biphasic dose-response curve: there was an
increase in amylase secretion with increasing dose of cerulein up to
the physiological dose of 1010 M, followed by a reduction
in secretion with supramaximal stimulation with up to 10
7
M cerulein. The biphasic dose-response curve was observed in acini even
after 24 h in culture (Fig. 2).
Production of MCP-1 and CINC by rat pancreatic acini as a function of time. In the absence of any stimulation, the levels of both MCP-1 and CINC in conditioned media increased with time up to 48 h, the latest time point analyzed (Fig. 3).
Production of MCP-1 and CINC by rat pancreatic acini in response to
cerulein treatment.
To investigate the effect of cerulein treatment on chemokine
production, pancreatic acini were incubated with cerulein for 30 min
(37°C). Conditioned media were then analyzed by ELISA. Treatment of
pancreatic acini with physiological doses of cerulein (1010 M) had little effect on MCP-1 synthesis, but higher
doses (10
7 M) of cerulein increased MCP-1 synthesis (Fig.
4A). After 24 h in
culture, treatment of acini with 10
7 M cerulein still
increased synthesis of MCP-1 (Fig. 4A). Synthesis of CINC,
however, was not affected after treatment with cerulein in both fresh
acini and acini after 24 h in culture (Fig. 4B).
NF-B activation by supramaximal stimulation with cerulein.
Detectable levels of NF-
B DNA binding were observed in nuclear
extracts from nontreated acini (Fig.
5A). An increase in NF-
B DNA binding was, however, observed in nuclear extracts from acini treated with a supramaximal dose of cerulein (Fig. 5A). The
addition to EMSA reactions of an excess of unlabeled competitor
oligonucleotide containing the NF-
B DNA binding sequence resulted in
a decrease in the binding of complexes arbitrarily designated
a, b, c, and d (Fig.
5B). These complexes were not affected by the addition of
unlabeled competitor oligonucleotide containing a mutated NF-
B DNA
binding sequence (Fig. 5B). Supershift assays with
antibodies against p65, p50, and c-rel revealed that
complexes b and c contained p65 whereas
complexes a, b, and d contained p50
(Fig. 5C). Addition of anti-c-Rel antibody to reaction mixes
had no effect on the pattern of binding (Fig. 5C).
Complex b may represent the classic NF-
B dimer p65-p50
and complex d the p50-p50 homodimer. Cerulein treatment
resulted in increases in complexes b and c but
not in complex d.
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Effect of NF-B inhibitors on MCP-1 and CINC production.
Experiments were then performed to examine the effect of two NF-
B
inhibitors, NAC and TPCK, on synthesis of MCP-1 and CINC by pancreatic
acini. Pretreatment of pancreatic acini with either antagonist blocked
both NF-
B activation and the upregulation of MCP-1 synthesis that
occurred in response to cerulein hyperstimulation in freshly isolated
acini or in acini after 24 h in culture (Fig. 6, A and C).
Neither inhibitor had any effect on basal production of MCP-1 from
nonstimulated acini. In parallel experiments, both NAC and TPCK
inhibited the cerulein-induced increase in NF-
B complexes
b and c (Fig. 6B). Neither inhibitor had any
effect on amylase secretion in response to different doses of cerulein (data not shown).
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Effect of TNF- on MCP-1 and CINC production.
To investigate the effect of TNF-
treatment on chemokine production,
pancreatic acini were incubated with TNF-
for 30 min (37°C).
Conditioned media were then analyzed by ELISA. Treatment of pancreatic
acini with TNF-
increased MCP-1 synthesis (Fig. 7). Synthesis of CINC, however, was not
affected after treatment with TNF-
(Fig. 7). Pretreatment of acini
with the NF-
B inhibitors NAC and TPCK blocked the upregulation
of MCP-1 synthesis in response to TNF-
(Fig.
8).
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Effect of thapsigargin and BAPTA-AM on MCP-1 and CINC synthesis.
To investigate the effect of the endoplasmic reticulum
Ca2+-ATPase inhibitor thapsigargin, which increases
intracellular Ca2+ levels, on chemokine production,
pancreatic acini were incubated with thapsigargin for 30 min (37°C).
Conditioned media were then analyzed by ELISA. Treatment of pancreatic
acini with thapsigargin increased MCP-1 synthesis in fresh acini or
after 24 h in culture. CINC production was not affected at
either time point (Fig. 9).
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DISCUSSION |
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Administration of supramaximal doses of the CCK analog cerulein to experimental animals leads to acute pancreatitis, which early on is associated with activation of trypsinogen, vacuole formation, and disrupted release of zymogen granules (16, 29, 33). Abnormalities of calcium signaling are thought to play a critical role in these events (33). Hyperstimulation of isolated pancreatic acini with CCK and its analog cerulein causes morphological changes that are similar to those seen in vivo and provides an ideal model with which to dissect the intra-acinar events in vitro. Whereas a physiological concentration of CCK does not cause intracellular trypsin activation, exposure to supramaximal CCK does have such an effect. This activation depends on a rise in intracellular Ca2+ concentration and can be blocked by the intracellular Ca2+ chelator BAPTA-AM (28, 33).
Most cells have the ability to express and synthesize chemokines, and
typically this occurs rapidly after cellular injury (1).
The profile of chemokines expressed at a site of injury is one of the
major factors that determine the nature of the subsequent leukocyte
infiltrate (1, 2). Inflammatory mediators involved in
acute pancreatitis, particularly those involved in the early stages of
an attack, may be derived directly from pancreatic acinar cells. For example, pancreatic acinar cells have been shown to produce and release TNF- and via specific receptors these cells respond (by apoptosis) to TNF-
(18).
Recent work in our laboratory (8) demonstrated expression
of MCP-1 on pancreatic acini in cerulein-induced pancreatitis in the
rat. Using the same model, Grady et al. (15) showed an increase in pancreatic MCP-1 mRNA (by Northern blot analysis) on
induction of acute pancreatitis, although the cellular origin of MCP-1
was not established. Two groups have reported production of MCP-1 by
isolated pancreatic acini (7, 34). In the study of Blinman
et al. (7), MCP-1 mRNA levels were upregulated in individual isolated acinar cells. Inhibitors of p38 mitogen-activated protein kinase (MAPK) and NF-B reduced levels of MCP-1 mRNA. In
contrast to isolated acini, no or very low MCP-1 expression was
detected in normal rat pancreas, suggesting thereby that activation of
p38 MAPK, activation of NF-
B, and synthesis of cytokines occur as a
result of removal of the pancreas from the animal and isolation of
acini (7). Our results confirm production of MCP-1, a CC chemokine, by isolated acini in short-term primary culture, and although physiological doses of cerulein did not affect its synthesis, treatment with supramaximal doses of cerulein increased MCP-1 synthesis.
The transcription factor NF-B is involved in the regulation of
several cytokines and chemokines that together have proinflammatory effects (13, 30). In nonstimulated cells NF-
B/Rel
proteins are sequestered in the cytoplasm in ternary complexes of
NF-
B homo- or heterodimers bound to specific inhibitor proteins
(I
Bs). After stimulation, I
B is degraded by proteasomes and
active NF-
B/Rel is translocated to the nucleus (13,
30). Cerulein hyperstimulation has been reported to cause
NF-
B activation in isolated acini and in pancreas in vivo (19,
20, 32).
We observed that the increase in MCP-1 production by isolated
pancreatic acini correlated with an increase in nuclear NF-B DNA
binding activity. Interestingly, not all of the observed
NF-
B-containing complexes were affected by cerulein
hyperstimulation. The quickly migrating complex d, which
contained p50 (Fig. 5C) and likely represents p50-p50
homodimer, was not altered by cerulein hyperstimulation; p50 homodimers
have been associated with transcription repression (10,
14). In contrast, the potently transactivating heterodimer p65-p50 (complex b; Fig. 5) exhibited increased DNA binding
activity. These data are consistent with the observed increase in MCP-1 production.
We further investigated the role of NF-B in cerulein-induced MCP-1
production by using two inhibitors of NF-
B activation, the
antioxidant NAC (24) and the proteasome inhibitor TPCK
(22). Reactive oxygen intermediates are produced and
released concurrently with NF-
B activation during the inflammatory
response (13), and antioxidants such as NAC are known to
be inhibitors of NF-
B activation (24). The importance
of reactive oxygen species in the early pathogenesis of acute
pancreatitis is well established (30). Proteasome
inhibition prevents activation of NF-
B by blocking the degradation
of the inhibitor protein I
B. Consequently, NF-
B is retained in
the cytoplasm in an inactive form. Pretreatment with either of the
NF-
B antagonists, NAC or TPCK, prevented both cerulein-induced
activation of NF-
B and cerulein-mediated production of MCP-1.
Treatment of pancreatic acini with thapsigargin, which increases cytosolic Ca2+ levels, leads to increased MCP-1 production. Pretreatment of isolated acini with the intracellular Ca2+ chelator BAPTA-AM prevented cerulein-mediated increase in MCP-1 synthesis, suggesting that the increase in cytosolic Ca2+ seen after cerulein hyperstimulation is a prerequisite for increased MCP-1 production.
TNF- has been shown to induce MCP-1 production in many cell types.
It is also an important inflammatory mediator in acute pancreatitis.
Treatment of isolated acini with TNF-
increased production of MCP-1.
This was NF-
B and Ca2+ dependent because pretreatment of
isolated acini with BAPTA-AM and NF-
B inhibitors inhibited
TNF-
-mediated production of MCP-1. There are thus similarities
between the regulation of chemokine production in response to cerulein
and TNF-
. Furthermore, TNF-
had no effect on CINC production by
isolated acini, reinforcing the concept of a differential regulation of
these two chemokines in pancreatic acinar cells.
Supraphysiological concentrations of CCK have recently been shown to
induce expression of mob-1, a CXC ELR-negative chemokine, in rat
pancreatic acini through the activation of the transcription factor
NF-B. CCK induced NF-
B nuclear translocation, and DNA binding was also blocked by BAPTA-AM (21). ELR-negative
CXC chemokines have little effect on neutrophils, and their profile of
activity resembles in many ways that of CC chemokines such as MCP-1.
For example, ELR-negative CXC chemokines, like CC chemokines, recruit
and activate lymphocytes and monocytes (25).
Production of chemokines, including CINC, by nonstimulated isolated
acini may in part be explained by the stress of isolation and culture
(7). Increased CINC mRNA levels, however, were demonstrated in pancreas after induction of acute pancreatitis by
cerulein hyperstimulation in vivo in rats (8, 19). We showed previously (6) that plasma levels of CINC are
elevated in acute pancreatitis and that a blocking anti-CINC antibody
reduces lung injury. Furthermore, we have shown that circulating levels of GRO-, the human homolog of CINC, are elevated in severe
pancreatitis (31). Together these findings suggest that
CINC is an important mediator in the inflammatory response in acute
pancreatitis. In our experiments, however, neither cerulein
hyperstimulation nor TNF-
treatment stimulated CINC production in
vitro from pancreatic acini. These results suggest that CINC production
in pancreatic acinar cells is regulated by a mechanism independent of
NF-
B or Ca2+.
This study demonstrates that MCP-1 and CINC are produced by isolated pancreatic acini and supports the idea that they may be involved in the early inflammatory response in acute pancreatitis. Furthermore, the differential regulation of CINC compared with MCP-1 suggests that there are fundamental differences in the regulation of chemokine synthesis within the acinar cell.
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ACKNOWLEDGEMENTS |
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This work was supported by Wellcome Trust Project Grant 003393.
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FOOTNOTES |
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10.1152/ajpgi.00031.2002
Address for reprint requests and other correspondence: M. Bhatia, Dept. of Pharmacology, National Univ. of Singapore, Faculty of Medicine, 10 Kent Ridge Crescent, Singapore 119260.
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 20 January 2001; accepted in final form 24 September 2001.
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