Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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Inflammatory mediators are involved in the early phase of acute
pancreatitis, but the cellular mechanisms responsible for their
generation within pancreatic cells are unknown. We examined the role of
nuclear factor-B (NF-
B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine
expression in pancreatic acinar cells in vitro. Supraphysiological, but
not physiological, concentrations of CCK-8 increased inhibitory
B
(I
B-
) degradation, NF-
B activation, and
mob-1 gene expression in isolated
pancreatic acinar cells. CCK-8-induced I
B-
degradation was
maximal within 1 h. Expression of
mob-1 was maximal within 2 h. Neither
bombesin nor carbachol significantly increased
mob-1 mRNA or induced I
B-
degradation. Thus the concentration, time, and secretagogue dependence
of mob-1 gene expression and I
B-
degradation were similar. Inhibition of NF-
B with pharmacological
agents or by adenovirus-mediated expression of the inhibitory protein
I
B-
also inhibited mob-1 gene
expression. These data indicate that the NF-
B signaling pathway is
required for CCK-8-mediated induction of
mob-1 chemokine expression in
pancreatic acinar cells. This supports the hypothesis that NF-
B
signaling is of central importance in the initiation of acute pancreatitis.
pancreas; pancreatitis; cytokines; adenovirus
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INTRODUCTION |
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THE CELLULAR MECHANISMS that initiate the development of acute pancreatitis are uncertain. It has been very difficult to investigate the cellular mechanisms involved in acute pancreatitis in humans because of its multifaceted clinical manifestations and the difficulty of early diagnosis. A number of animal models of experimental pancreatitis have been developed to investigate the biochemical, morphological, and pathophysiological changes that accompany this disease (15, 17, 41). Although progress in understanding the pathophysiology of acute pancreatitis has been achieved using these models, the cellular mechanisms and factors that initiate the disease remain unclear, partly because cellular mechanisms are obscured by the complications of events outside the pancreas in vivo. In vitro models offer advantages for mechanistic studies.
The development of acute pancreatitis is often considered as consisting
of two phases: an early phase involving pancreatic acinar cell damage
and a secondary phase involving an inflammatory response. Mechanisms
that lead to acinar cell damage observed during acute pancreatitis have
been well studied and are thought to include the intracellular
activation of digestive enzymes (41). In contrast, the cellular
mechanisms responsible for the initiation of the inflammatory response
are less well understood. Once initiated, the inflammatory response may
spread to other organs, particularly the lungs, and is the primary
cause of mortality associated with the disease (42). Evidence is
accumulating that an important event in the evolution of the
inflammatory phase of acute pancreatitis is release of endogenous
inflammatory mediators, such as cytokines and chemokines (23). The
serum levels of interleukin-6, interleukin-8, tumor necrosis
factor-, and platelet-activating factor are known to increase during
acute pancreatitis and are well correlated with the severity of the
disease (18, 22, 32, 45). The source of these cytokines has not been
clear, and it is likely that many of these inflammatory regulators are
released from infiltrating immune cells as a late response (12).
However, it has recently been suggested that cytokines may originate
within the pancreas (19, 33). We found that acinar cells themselves
express chemokines, a class of cytokines known for their
chemoattractive functions, early in the course of experimental acute
pancreatitis (16).
Chemokines attract and activate inflammatory cells (29, 38). Therefore,
chemokines are ideally suited to play a critical role in the earliest
initiating events in pancreatitis. We found a dramatic increase in the
expression of mob-1, a member of the -chemokine (C-X-C) family, and
mcp-1, a member of the
-chemokine (C-C) family, in pancreatic acinar cells early in the course of experimental acute pancreatitis (16). These observations suggested that
general cellular mechanisms leading to the expression of a variety of
chemokine genes were likely activated in an early phase of acute
pancreatitis. However, little is known concerning the cellular
mechanisms involved in chemokine gene expression in pancreatic acinar cells.
In the present study we investigated the cellular mechanisms involved
in cholecystokinin octapeptide (CCK-8)-induced chemokine gene
expression in pancreatic acinar cells in the absence of the confounding
factors existing in vivo by utilizing an in vitro preparation of
isolated pancreatic acini. Expression of chemokines and other cytokines
is widely regulated by the transcription factor nuclear factor-B
(NF-
B) (4). Recently, it has been reported that cerulein
hyperstimulation activates NF-
B in vivo in a manner that correlates
with the development of pancreatitis (20, 43). In the present study we
analyzed the relationship between the activation of NF-
B and the
expression of mob-1 in vitro. We also utilized several independent methods to inhibit NF-
B activation, including the overexpression of the inhibitory protein I
B-
, using
adenovirus-based gene transfer. Our results indicate that mob-1 chemokine gene expression is an
early and specific response to CCK-8 hyperstimulation and that NF-
B
activation is required for the regulation of this chemokine gene in
pancreatic acinar cells.
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MATERIALS AND METHODS |
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Materials.
Chromatographically purified collagenase was purchased from Worthington
Biochemical (Freehold, NJ). Soybean trypsin inhibitor (SBTI),
carbachol, bombesin, -mercaptoethanol, phenylmethylsulfonyl fluoride, sodium orthovanadate, HEPES, glutamine, and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma Chemical (St. Louis,
MO). CCK-8 was purchased from Research Plus (Bayonne, NJ). Enhanced
chemiluminescence detection reagents,
[
-32P]ATP,
d-[
-32P]CTP, and
goat anti-rabbit IgG horseradish peroxidase conjugate were from
Amersham (Arlington Heights, IL). The electrophoretic mobility shift
assay (EMSA) systems kit was obtained from Promega (Madison, WI). The
rabbit polyclonal antibodies to I
B-
and NF-
B p65, p50, and
c-Rel subunits were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Eagle's minimum essential amino acids, guanidine thiocyanate, and
agarose were purchased from GIBCO BRL Life Technologies (Gaithersburg, MD).
Pancreatic acini isolation and treatments. Pancreatic acini were prepared by a modification of previous methods (27). Briefly, pancreata from male Wistar rats were quickly (<10 min) injected with collagenase (100 U/ml) in a Krebs-Henseleit bicarbonate medium to which SBTI (0.1 mg/ml) and minimal essential amino acids had been added. Injected pancreata were then minced into three to five pieces and incubated at 37°C for 45-50 min with shaking (120 cycles/min). Acini were then dispersed using an electric pipette aid gently triturating the pancreas through polypropylene pipettes with decreasing orifice (3.0, 2.4, and 1.2 mm). After filtration through a 150-µm nylon mesh, acini were purified by centrifugation at 10 g for 5 min in a solution containing 4% BSA and resuspended in HEPES-buffered Ringer solution (pH 7.5) supplemented with 0.2% glucose, Eagle's minimum essential amino acids, 2 mM glutamine, SBTI (0.1 mg/ml), and 0.5% BSA. The dispersed acini were aliquoted and treated with various hormones and pharmacological agents at indicated concentrations in HEPES-buffered Ringer solution for specified times in 100-mm tissue culture dishes. All treatments and incubations were conducted in a cell culture incubator at 37°C in a humidified atmosphere.
Adenoviral infection.
An adenoviral vector with a cytomegalovirus promoter driving expression
of a full-length IB-
cDNA modified by the addition of a nuclear
translocation sequence (AdI
B-
)
was a kind gift of Dr. J. Anrather (Beth Israel Deaconess Medical
Center, Boston, MA). A previously described adenovirus bearing the
bacterial
-galactosidase gene (AdLacZ) (3)
obtained from Dr. B.J. Roessler (University of Michigan, Ann Arbor, MI)
was utilized as a control. Acini prepared as described above were
infected with this adenovirus, as described previously (31). Adenovirus
titers ranged from 107 to
109 plaque-forming units/mg acinar
protein (multiplicity of infection = 10-1,000) as indicated. Acini
were then incubated for 8 or 20 h before the addition of CCK-8 for
indicated times.
Isolation of RNA and analysis of mob-1 mRNA expression.
Total RNA was isolated by a modified acid
guanidinium-thiocyanate-phenol-chloroform extraction (8). Briefly,
after incubation with or without indicated agonists, pancreatic acini
pellets were homogenized in 3 ml of 4 M guanidine thiocyanate buffer
containing 8% -mercaptoethanol with use of a Polytron (Brinkman
Instruments, Westbury, NY). Phenol-chloroform extraction was performed
immediately, and the aqueous phase was precipitated with isopropanol.
The pellets were dissolved into 4 M guanidine thiocyanate buffer, and
the RNA was reextracted with phenol-chloroform and precipitated with isopropanol at
20°C overnight. RNA was quantitated
spectrophotometrically, and 25 µg from each sample were
electrophoresed in 1% agarose and 2.2 M formaldehyde gels in 1×
MOPS buffer and transferred to a Hybond
N+ membrane (Amersham).
Mob-1 mRNA was detected using a
full-length 1.2-kb cDNA of rat mob-1
(gift from Dr. P. Liang, Vanderbilt University, Nashville, TN).
Membranes were hybridized at high stringency using QuickHyb solution
(Stratagene, La Jolla, CA) with the
d-[
-32P]CTP-labeled
mob-1 probe at 68°C for 2 h. After
hybridization the membranes were exposed to a B-1 phosphor-imaging
screen and visualized using the GS-250 Molecular Imaging System
(Bio-Rad Laboratories, Richmond, CA). Ethidium bromide staining of
total RNA was used as a loading control.
IB-
immunoblot analysis.
Dispersed acini were treated as described in Figs. 1-8. The
treatments were terminated by washing the acini with ice-cold PBS containing 1 mM
Na3VO4.
The pellets were lysed by sonication for 5 s in a solution containing
50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and a
protease inhibitor cocktail containing 10 µg/ml each of aprotinin,
leupeptin, and pepstatin. The samples were then set on ice for 15 min
and centrifuged at 14,000 g for 15 min
at 4°C. The supernatant was removed as whole cell lysate and assayed for protein by protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (20 µg) were resolved by SDS-PAGE and
transferred to a nitrocellulose membrane. I
B immunoblot analysis was
performed as described previously (21) and visualized with enhanced
chemiluminescence reagent on film or screen.
Preparations of nuclear extracts. Nuclear extracts were prepared using a modified version of the method of Maire et al. (28). After the different treatments described in the experimental design, pancreatic acini were collected by brief centrifugation and washed with ice-cold PBS containing 1 mM EDTA. The pellets were then resuspended in 0.5 ml of homogenization buffer containing 10 mM HEPES (pH 7.9), 2 M sucrose, 10% glycerol (vol/vol), 25 mM KCl, 150 mM spermine, 500 mM spermidine, and 2 mM EDTA, to which 1 mM dithiothreitol (DTT) and the protease inhibitor cocktail described above were added before use. Cells were homogenized with a motor-driven pestle for 5-10 strokes on ice. Nuclei were collected by centrifugation at 62,000 g for 30 min at 4°C and washed with 1 ml of PBS containing 1 mM EDTA, then centrifuged at 14,000 g for 5 min at 4°C. The nuclei were resuspended in an appropriate volume (~100 µl) of ice-cold high-salt buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 0.42 M NaCl, 100 mM KCl, 3 mM MgCl2, and 0.1 mM EDTA, to which DTT and the protease inhibitor cocktail described above were added. The nuclear suspension was incubated on ice for 15-30 min with intermittent mixing and centrifuged at 14,000 g for 5 min at 4°C. Protein concentration in the nuclear extract was determined using the Bio-Rad protein assay reagent. The same procedure was also utilized for preparation of nuclear extract from untreated pancreatic tissue.
EMSA.
Aliquots of nuclear extract with equal amounts of protein (6-12
µg) were utilized in 10-µl reactions in a buffer containing 10 mM
HEPES (pH 7.9), 10% glycerol (vol/vol), 1 mM DTT, 1 µg of poly(dI-dC), and 5 µg of nuclease-free BSA. The binding reaction was
started by addition of 10,000 cpm of the 22-bp oligonucleotide 5'-AGT TGA C AGG
C-3' containing the NF-
B consensus sequence (underlined) or
the 22-bp oligonucleotide 5'-TGT CGA ATG CAA ATC ACT AGA
A-3' containing the OCT1 sequence (Promega) that had been labeled
with [
-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. The reaction was allowed to
proceed for 30 min at room temperature. For cold competition
experiments, unlabeled NF-
B oligonucleotide or OCT1 oligonucleotide
as a nonspecific competitor (300×) was added to the binding
reaction 5 min before the addition of the radiolabeled probe. For
antibody supershift assays, 2 µl of specific antibodies to NF-
B
protein subunits p65, p50, and c-Rel were incubated with nuclear
extracts for 1 h at room temperature before the addition of labeled
probe. All reaction mixtures were subjected to PAGE on 4.5% gel in
0.5× Tris base-EDTA-boric acid buffer at 200 V. Gels were dried
and directly exposed to B-1 phosphor-imaging screen, as indicated
previously for Northern blots.
NF-B immunoblotting analysis.
Nuclear extracts were prepared as described above. Aliquots with equal
amounts of nuclear protein from treated acinar cells or intact
pancreata were subjected to SDS-PAGE on a 10% gel and transferred onto
a nitrocellulose membrane. The NF-
B protein was detected by a
specific antibody against the p65 subunit of NF-
B.
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RESULTS |
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CCK-8 activates NF-B in pancreatic acini in vitro.
On the basis of the known role of NF-
B in inducing the expression of
a wide variety of inflammatory mediators, we hypothesized that NF-
B
might mediate the expression of the chemokine
mob-1 stimulated by supraphysiological
concentrations of CCK-8 in the pancreas. Activation of NF-
B involves
the phosphorylation and degradation of the inhibitory protein I
B-
and the nuclear translocation and DNA binding of NF-
B. To determine
the ability of CCK-8 to activate NF-
B in pancreatic acini in vitro,
we examined the effects of CCK-8 on nuclear translocation of the
NF-
B p65 subunit and on NF-
B DNA binding. Only low levels of
NF-
B consensus site binding were observed in EMSAs with use of
nuclear extracts from untreated pancreatic tissue (Fig.
1A, lane
2) and control acini (Fig.
1A, lane 3), indicating that the
procedure for isolation of acini did not activate this signaling
pathway. Likewise, only low levels of p65 NF-
B were detected in
Western blots of nuclear protein extracts from control acini (Fig.
2). Treatment with CCK-8 (100 nM) for 1 h
increased the amount of p65 subunit detected in the nuclear fraction by
5.3 ± 0.6-fold (n = 6; Fig. 2) and led to a marked increase (4.9 ± 0.9-fold,
n = 6) in NF-
B consensus site
binding (Fig. 1A, lane 4). These
data indicate that treatment of isolated pancreatic acini with
supraphysiological concentrations of CCK-8 activates NF-
B, as has
recently been reported by others (20, 43).
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CCK-8 induces chemokine gene expression and
IB-
degradation in a dose- and
time-dependent manner in dispersed rat pancreatic acini.
Similar to what is observed with other NF-
B activators, CCK-8
activation of NF-
B was associated with a decrease in cytoplasmic levels of the inhibitory component I
B-
(Fig.
3). To define the concentration and time
dependence of CCK-8-mediated NF-
B activation and
mob-1 gene expression, we analyzed
samples of treated acini for I
B-
protein levels and
mob-1 mRNA. There was minimal
I
B-
degradation (Fig. 3A) and
mob-1 gene expression (Fig.
3B) in freshly isolated pancreatic
acini (lane 2). This was similar to
observations in undisturbed pancreatic tissue (Fig. 3,
A and B, lane
1). After treatment with CCK-8 (100 nM), I
B-
was degraded, as evidenced by loss of the I
B-
band in Western
blots (16 ± 6% of control acini,
n = 9), and
mob-1 gene expression was induced, as
evidenced by an increased amount of
mob-1 mRNA in Northern blot analysis (4.5 ± 0.6-fold, n = 9).
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Bombesin and carbachol do not cause
IB-
degradation or mob-1 expression.
To determine the specificity of the ability of CCK-8 to degrade
I
B-
and induce mob-1 gene
expression, we tested the effects of other secretagogues (Fig.
5). After treatment of dispersed pancreatic
acini with supraphysiological concentrations of bombesin (100 nM) or
carbachol (1 mM) for 1 h in vitro, most of I
B-
protein [92.5 ± 14.4% (n = 4) and
87.5 ± 16.5% (n = 4) of
control, respectively] remained undegraded. There was also little
or no increase in mob-1 mRNA levels
induced by treatment with bombesin or carbachol for 2 h. These results
support the hypothesis that mob-1 gene
expression is associated with pancreatitis and not simply secretagogue
hyperstimulation.
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PDTC pretreatment inhibits NF-B activation and
blocks induction of mob-1 chemokine expression in isolated pancreatic
acini.
An inhibitor for NF-
B activation, PDTC, was utilized to explore the
relationship between CCK-8-induced NF-
B activation and mob-1 gene expression. PDTC
pretreatment prevented the degradation of I
B-
induced by
supraphysiological concentrations of CCK-8, as evidenced
by Western blotting (Fig.
6A), and
the increase of mob-1 mRNA, as
evidenced by Northern blot analysis of total RNA isolated from acini of
the same experiment (Fig. 6C). PDTC
pretreatment also blocked NF-
B p65 subunit translocation to the
nucleus (data not shown) and the appearance of a NF-
B consensus site
binding band (Fig. 6B). Thus PDTC
blocked NF-
B activation and mob-1
gene expression stimulated by supraphysiological concentrations of CCK-8, suggesting a role for NF-
B in CCK-8-mediated
mob-1 gene expression.
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Adenovirus-mediated expression of
IB-
inhibits CCK-8-induced mob-1 gene
expression.
To verify the role of NF-
B in mob-1
gene expression, a more specific approach using adenovirus-mediated
gene transfer of the inhibitory I
B-
protein was utilized. In this
approach, acini were infected with an adenovirus bearing an
I
B-
gene modified to localize to
the cell nucleus (44). In previous studies, adenovirus-mediated gene
transfer to isolated acinar cells was found to be highly efficient and
titer dependent (31, 34). To analyze the effects of adenoviral
infection on I
B-
levels, we determined the effects of infection
with various titers of a control adenovirus bearing the
-galactosidase gene and those of
infection with adenovirus bearing the
I
B-
gene (Fig.
7). Infection with the control virus led to
a decrease in basal levels of acinar I
B-
, and these levels were
further decreased by treatment of acini with CCK-8 (Fig. 7A). In contrast, infection with
adenovirus bearing I
B-
led to a titer-dependent increase in the
levels of I
B-
. At lower titers, treatment with supraphysiological
concentrations of CCK-8 continued to cause a significant
decrease in I
B-
levels. However, at higher titers of adenovirus
bearing I
B-
, no effect of CCK-8 on I
B-
levels was noted
(Fig. 7B). Thus, after adenoviral
delivery of the modified I
B-
gene, CCK-8 was no longer able to cause a major decrease in whole cell
I
B-
levels.
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DISCUSSION |
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We recently demonstrated that chemokine expression occurs early in the course of acute pancreatitis and originates from acinar cells themselves (16). We showed a correlation between the expression of two chemokine genes, mob-1 and mcp-1, and the manifestations of experimental acute pancreatitis in two separate models, secretagogue hyperstimulation and retrograde bile infusion. In the secretagogue hyperstimulation model, mob-1 gene expression and the severity of pancreatitis shared secretagogue and concentration dependence. We have also observed an association of mob-1 gene expression and the severity of pancreatitis in an arginine model of acute pancreatitis (Han, Tashiro, Williams, and Logsdon; unpublished observation). Recently, it has been shown that systemic manifestations of acute pancreatitis, especially the associated lung inflammation, were significantly reduced in animals lacking one of the chemokine receptors (39). Taken together, these observations and the known role of chemokines in other inflammatory diseases suggest that chemokines are likely important in the initiation of the inflammatory component of acute pancreatitis. However, the cellular mechanisms involved in pancreatic acinar cell chemokine expression have not been elucidated.
The transcription factor NF-B is considered of central importance in
inflammation for its ability to induce expression of a variety of genes
that amplify and perpetuate inflammatory responses. That NF-
B was
activated during cerulein-induced pancreatitis was suggested by the
ability of inhibitors of NF-
B to block
mob-1 gene expression and early
parameters of pancreatitis (16). More recently, activation of NF-
B
during experimental pancreatitis has been reported by several groups
(11, 20, 43). NF-
B regulates several families of rapid-response and
inflammatory genes, including those for chemokines, cytokines,
inflammatory enzymes, adhesive molecules, and growth factors (1, 4,
14). Therefore, we hypothesized that NF-
B would be involved in the regulation of acinar cell mob-1 gene
expression. Consensus NF-
B binding sites have been identified within
the promoter region of the mob-1 gene,
but direct regulation by NF-
B has not been shown for
mob-1.
The activation of NF-B generally involves the phosphorylation,
dissociation, and proteolytic degradation of inhibitory proteins of the
I
B family. I
B proteins function to mask the nuclear localization signals found in the NF-
B subunits that prevent the NF-
B dimers from translocating to the nucleus in unstimulated cells (40). Therefore, in some experiments we utilized I
B-
protein levels as
an indirect marker for NF-
B activation. In the present study conducted in vitro, I
B-
levels were rapidly reduced by CCK-8 within 10 min, and no recovery was observed for
4 h. This is similar
to in vivo observations reported by Steinle et al. (43) but differs
from the in vivo observations of Gukovsky et al. (20), who observed a
rapid recovery of I
B-
levels within 3 h. Differences in I
B-
recovery between in vitro and in vivo models might be explained by the
influence of infiltrating inflammatory cells at later times in the in
vivo situation. However, the explanation for the observed differences
in I
B-
recovery between the in vivo studies is unknown.
We found that CCK-8 treatment of dispersed acinar cells led to a dose-
and time-dependent decrease in IB-
protein. Significant decreases
of I
B-
levels were only observed in acinar cells stimulated with
supraphysiological concentrations of CCK-8, correlating with the known
concentration dependence of CCK-8 on pancreatitis in vivo. Furthermore,
the concentration dependence of I
B-
degradation and
mob-1 mRNA expression were identical.
We also observed a direct correlation between I
B-
degradation and
NF-
B nuclear translocation and DNA binding. The correlation between
CCK-8-induced I
B-
protein degradation and
mob-1 gene expression in the same
preparation supports the hypothesis that the activation of NF-
B is
involved in the regulation of mob-1 expression.
In contrast to the effects of CCK-8, neither IB-
degradation nor
mob-1 expression was induced by the
secretagogues bombesin and carbachol. Bombesin and carbachol are
equivalent to CCK-8 in terms of their abilities to stimulate acinar
cell secretion. However, bombesin does not cause pancreatitis in this
model (35). Whether carbachol administration can induce pancreatitis in
the rat is controversial. Bilchik et al. (5) found that administration of carbachol, a cholinergic agonist, produced a mild form of
pancreatitis that was partially blocked by a CCK-8 receptor antagonist.
Robert et al. (37) found that carbachol treatment did not induce
pancreatitis, even when rats were treated with toxic concentrations. We
were unable to induce pancreatitis in the rat by intraperitoneal
administration of carbachol (unpublished observation). Others have
shown that the CCK analog JMV-180, which is fully efficacious as a
secretagogue but does not cause pancreatitis, also does not activate
NF-
B when administered at high concentrations to rats (20). Thus there appears to be an excellent correlation between the ability of
secretagogues to induce pancreatitis and their abilities to activate
NF-
B and trigger expression of proinflammatory mediators.
To directly demonstrate a role for NF-B in the induction of
mob-1 expression in pancreatic acinar
cells, we utilized a variety of methods to inhibit NF-
B activation.
Previously, we reported that PDTC, a known inhibitor of NF-
B
activation in a variety of cell models (40), inhibited
mob-1 gene expression in vivo (16). In
the present study we found that PDTC blocked NF-
B activation in
acinar cells in vitro. Recently, it has been reported that PDTC and
N-acetylcysteine, another known
inhibitor of NF-
B activation, inhibited the ability of cerulein
hyperstimulation to activate NF-
B in vivo (20, 43). However, PDTC is
also known to be an antioxidant and an iron chelator (6) and may have
nonspecific actions on cell function. Likewise,
N-acetylcysteine is a powerful
antioxidant with multiple effects on cell function. These antioxidants
have recently been shown to have posttranscriptional effects that do
not affect NF-
B activity (7). Therefore, in the present study, to
more directly demonstrate a role for NF-
B in
mob-1 gene expression, we utilized a
molecular approach.
Adenoviral vectors have recently been shown to be a highly efficient
method for gene transfer to isolated acini (31, 34). These studies have
shown that infection of acini with adenovirus bearing
-galactosidase has no obvious deleterious
effects on acini, as assessed by effects on lactate dehydrogenase
release or the ability of CCK-8 to stimulate increases in intracellular Ca2+ or amylase release or to
activate MAP kinases. However, the data in the present study suggest
that adenoviral infection may acutely activate NF-
B and stimulate
mob-1 expression in pancreatic acinar cells. It has previously been reported that adenoviral infection at
high titer stimulates NF-
B expression in other cells, including human vascular smooth muscle cells (9) and hepatocytes (25). This
effect may account for some of the inflammation previously noted when
adenovirus was utilized to deliver genes to the pancreas in vivo (36).
In contrast to the effects of infection with a control adenovirus
bearing -galactosidase, infection with
adenovirus bearing I
B-
did
not raise basal levels of mob-1
expression. Furthermore, adenovirus-mediated expression of I
B-
blocked the effects of supraphysiological concentrations of CCK-8 on
mob-1 expression. This inhibitory
effect was noted within 8 h of adenoviral infection, a time at which no
effects were noted on other CCK-8-mediated effects, including the
stimulation of Ca2+ release or
amylase secretion (data not shown). Expression of I
B-
acts as a
very specific inhibitor of NF-
B activation and has been widely
utilized as a means of determining the effects of NF-
B activation on
cellular activation and gene expression (14, 26, 30, 44). The
adenovirally delivered I
B-
utilized in this study was modified by
the addition of a nuclear translocation signal, such that this molecule
localizes at least in part to the cell nucleus (44). I
B-
has a
high affinity for the NF-
B family of proteins and can prevent their
binding to DNA (10). This construct was previously shown to block
NF-
B activation of cytokines and cell adhesion molecules in
endothelial cells without affecting gene expression mediated by other
transcription factors or interfering with other cellular functions not
linked to NF-
B activation (44). We observed no effect of this virus on CCK-8-mediated Ca2+ signaling
or secretion from isolated acini after 8 or 20 h (data not shown). Thus
the present data strongly support the hypothesis that NF-
B
activation is directly involved in the induction of mob-1 chemokine expression by CCK-8 in
pancreatic acinar cells.
Much is yet to be discovered about the identities and roles of inflammatory mediators involved in the local and systemic responses that occur during acute pancreatitis. Significant differences exist between rodents and humans in terms of the cytokines/chemokines produced and the specific roles of proinflammatory molecules. Mob-1 is a chemokine found in the rat that has significant structural homology with the human chemokine IP-10. However, whether these two molecules function in a homologous manner is unknown. Chemokines are small secreted molecules that are chemotactic for inflammatory cells and are typically induced and released at the site of injury (2, 38). According to the evidence from clinical and experimental studies, it has recently come to be well accepted that inflammatory mediators and cells are involved in the development of acute pancreatitis. The levels of several cytokines have been reported to be increased in serum of the patients with acute pancreatitis (18, 22, 32, 45). However, cytokines are a general feature of the inflammatory response that leads to a continuing infiltration of activated leukocytes and secondary cascade of cytokines. The mechanisms and proximal mediators underlying the inflammatory response in pancreatitis remain obscure. Thus it is necessary to distinguish between the secondary effects of cytokines released from infiltrated leukocytes and those initially released from damaged acinar cells.
Our results from isolated rat pancreatic acinar cells demonstrate that
injured acinar cells themselves are the source of chemokines. Because
they originate from acinar cells and are known to initiate leukocyte
infiltration and activation in other tissues, it is highly likely that
chemokines may be an important initiating signal for the development of
pancreatitis. Chemokines can also be released into the general
circulation and can influence cells at distant sites. It was recently
reported that the inflammatory response often observed in the lungs of
animals during acute pancreatitis was absent in animals in which the
CCR1 chemokine receptor had been knocked out by homologous
recombination (13). The CCR1 receptor is one of several receptors that
recognize -chemokines. The source and identity of the chemokines
that activate this receptor in the lungs during acute pancreatitis are
unknown, but it is likely that chemokines released from the pancreas
itself may be involved.
The present data indicate that NF-B activation is critical for the
expression of a proinflammatory mediator in rat pancreatic acinar
cells. It seems highly likely that these same cellular mechanisms are
important for the inflammatory response in human pancreatitis. Thus
these observations may be relevant to new approaches to intervention
and therapy for acute pancreatitis.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. A. Williams, B. Nicke, and D. Simeone for critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52067 and University of Michigan Gastrointestinal Peptide Center Grant DK-34933.
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 and other correspondence: C. Logsdon, Dept. of Physiology, Box 0622, University of Michigan, 7710 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622 (E-mail: clogsdon{at}umich.edu).
Received 29 January 1999; accepted in final form 5 April 1999.
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