Departments of 1 Medicine and 2 Surgery, Veterans Affairs Medical Center, West Los Angeles and University of California, Los Angeles, California 90073
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inflammation and cell death are critical to
pathogenesis of acute pancreatitis. Here we show that transcription
factor nuclear factor-B (NF-
B), which regulates these processes,
is activated and plays a role in rat cerulein pancreatitis. NF-
B was
strongly activated in the pancreas within 30 min of cerulein infusion; a second phase of NF-
B activation was prominent at 3-6 h. This biphasic kinetics could result from observed transient degradation of
the inhibitory protein I
B
and slower but sustained degradation of
I
B
. The hormone also caused NF-
B translocation and I
B
degradation in vitro in dispersed pancreatic acini. Both p65/p50 and
p50/p50, but not c-Rel, NF-
B complexes were manifest in pancreatitis
and in isolated acini. Coinfusion of CCK JMV-180, which abolishes pancreatitis, prevented cerulein-induced NF-
B activation. The second
but not early phase of NF-
B activation was inhibited by a
neutralizing tumor necrosis factor-
antibody. Antioxidant
N-acetylcysteine (NAC) blocked NF-
B
activation and significantly improved parameters of pancreatitis. In
particular, NAC inhibited intrapancreatic trypsin activation and mRNA
expression of cytokines interleukin-6 and KC, which were dramatically
induced by cerulein. The results suggest that NF-
B activation is an
important early event that may contribute to inflammatory and cell
death responses in acute pancreatitis.
pancreatic acinar cell; interleukin-6; KC; IB
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DESPITE CONSIDERABLE progress in understanding pathophysiology of pancreatitis, the mechanisms of the development of this disease remain obscure (52). A number of animal models of experimental pancreatitis have been developed and show biochemical, morphological, and pathophysiological similarities to various aspects of human pancreatitis (14, 31, 51, 54). One of the best-characterized and widely studied experimental models involves administration of high doses of cerulein, a CCK analog (14, 54). Doses of CCK or cerulein beyond those that cause maximal pancreatic secretion of amylase and lipase (26, 46) result in pancreatitis characterized by dysregulation of production and secretion of digestive enzymes, in particular, inhibition of pancreatic secretion and elevation of their serum levels, cytoplasmic vacuolization and death of acinar cells, edema formation, and infiltration of inflammatory cells into the pancreas (14, 31, 44, 51, 54).
Over the past several years, evidence has been accumulating on the
involvement of inflammatory mediators, such as cytokines-chemokines interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor- (TNF-
), and platelet-activating factor (PAF), in the development of
pancreatitis (8, 12, 15-17, 19, 25, 36-39, 45, 57). Systemic
levels of IL-6 and IL-8 increase in patients with acute pancreatitis and correlate with the severity of the disease (12, 16, 17, 25, 36). In
experimental pancreatitis, the levels of PAF, IL-1
, and TNF-
are
elevated not only in blood but also in the pancreas, and their blockade
attenuates the disease (8, 15, 19, 36-39, 45, 57). Furthermore, we
found recently that the pancreatic acinar cell itself is capable of
producing and releasing TNF-
(19). Although cytokines have been
implicated in the development and clinical course of pancreatitis,
their source, mechanism of regulation, and role at the early stages of
the disease remain obscure.
A key regulator of cytokine induction is the pleiotropic transcription
factor nuclear factor-B (NF-
B). NF-
B represents a family of
proteins sharing the Rel homology domain, which bind to DNA as homo- or
heterodimers, and activate a multitude of cellular stress-related and
early response genes such as the genes for cytokines, growth factors,
adhesion molecules, and acute phase proteins (reviews in 3, 32, 50, 55). Unlike most transcription factors, NF-
B is kept silent in the
cytoplasm via interaction with inhibitory proteins of I
B family
(I
B
, I
B
, Bcl-3, etc.) that masks the nuclear localization
signal. NF-
B is activated by a variety of agents ranging from
cell-damaging physical factors and viruses to mitogens and cytokines.
On activation I
B proteins become hyperphosphorylated and
proteolytically degraded, thus allowing NF-
B dimers to rapidly
translocate into the nucleus where they bind to DNA sites containing
B motifs. The selectivity of binding is controlled, at least
partially, by distinct protein subunits of NF-
B, such as p65 (RelA),
p50, and c-Rel (32, 50).
The two most abundant members of IB family, I
B
and I
B
,
are differently regulated by NF-
B itself (55). The gene encoding I
B
contains functional NF-
B sites and can be transcriptionally activated by NF-
B, which leads to rapid resynthesis of I
B
and blockade of NF-
B nuclear translocation (1, 24, 53). In contrast to
I
B
, no
B motifs have been found in the gene for I
B
, and
NF-
B does not regulate I
B
synthesis (24, 27, 53).
Apart from phosphorylation, the oxidative state of the cell has been
shown to play a role in NF-B activation (11, 42, 47, 48, 50, 55).
Several antioxidants, such as
N-acetylcysteine (NAC), block
activation of NF-
B by a number of inducers, indicating the
involvement of reactive oxygen intermediates (11, 42, 47, 55).
Little is known about NF-B in the pancreas. NF-
B activation plays
a central role in regulating the expression of genes involved in
inflammation, cell injury, and cell death. These processes mediate the
development of acute pancreatitis; however, apart from limited
observations (10, 20-22, 35), the role of NF-
B in pancreatitis
has not been studied.
The purpose of the present study was to determine if NF-B is
activated and plays a role in hormone-induced experimental
pancreatitis. We found that 1) in
rat cerulein pancreatitis NF-
B was rapidly and strongly activated
through degradation of both I
B
and I
B
; 2) cerulein-induced NF-
B
activation was prevented by coinfusion of the CCK analog JMV-180, a
treatment that abolishes pancreatitis; 3) cerulein directly activated
NF-
B through degradation of I
B in isolated pancreatic acini;
4) blockade of NF-
B activation by
NAC improved the parameters of the disease; and
5) the expression of
NF-
B-regulated genes for cytokines IL-6 and KC (murine analog of
chemokines IL-8/GRO-
) was dramatically induced in cerulein pancreatitis and was inhibited by NF-
B blockade. Thus
NF-
B activation is an early event that may have an important part in
the development of hormone-induced pancreatitis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental model of pancreatitis.
We randomized male Sprague-Dawley rats (Harlan, Madison, WI), weighing
270-320 g, into several groups in two categories (A and
B). The rats in category A
received continuous intravenous infusion of 5 µg · kg1 · h
1
cerulein (in physiological saline solution plus 30 U/l heparin) for a
period of up to 6 h at a rate of 0.6 ml/h. Rats in the main group were
infused with cerulein only; rats in other groups received, in addition
to cerulein, the CCK analog JMV-180, the antioxidant NAC, or a
neutralizing TNF-
antibody. CCK JMV-180 was infused at 5 mg · kg
1 · h
1
in combination with cerulein or alone. NAC was given in various regimens; most of the data were obtained on rats that were given an
intravenous bolus of 200 mg/kg NAC 2 h before cerulein treatment followed by continuous infusion of 25 mg · kg
1 · h
1
NAC for the duration of the experiment. Thus a 6-h cerulein-infused rat
received a total of 400 mg/kg (2.4 mmol/kg) NAC. Polyclonal rabbit
anti-mouse neutralizing TNF-
antibody (Genzyme, Cambridge, MA) was
given at 1:100 dilution as an intravenous bolus (10 µl diluted in 990 µl sterile PBS). Category B
consisted of rats used as controls in each group, which instead of
cerulein received an infusion of physiological saline solution plus 30 U/l heparin at the same rate.
Isolation of dispersed pancreatic acini. Dispersed pancreatic acini from rats were prepared using a previously published collagenase digestion method (41). To culture acinar cells, dispersed pancreatic acini were washed and resuspended in medium 199 supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 0.5% BSA. Cells were plated at a concentration of 5 × 105/ml in 25-ml tissue culture flasks and incubated with or without 0.7 µM cerulein for indicated times in a 5% CO2-humidified atmosphere at 37°C.
Preparation of nuclear extracts.
Nuclear protein extracts were prepared essentially as described by
Dignam et al. (9). A 150- to 200-mg portion from the splenic area of
the gland or a sample of isolated pancreatic acinar cells was rinsed in
ice-cold PBS and lysed on ice in the hypotonic buffer
A (9) by 20 or 5 strokes, respectively, in a glass
Dounce homogenizer. Just before use, buffer
A was supplemented with phenylmethylsulfonyl fluoride
(PMSF) and dithiothreitol (DTT) to the final concentration of 1 mM each
and with the protease inhibitor cocktail containing 5 µg/ml each of
pepstatin, leupeptin, chymostatin, antipain, and aprotinin. The
homogenate was left on ice for 15-20 min, and then Nonidet P-40
was added to the final concentration of 0.3-0.4% (vol/vol), and
the samples were briefly vortexed and incubated on ice for an
additional 1-2 min. Crude nuclear pellet was collected by
centrifugation of the lysed tissue or cell samples for 30 s in a
Microfuge. The supernatant (cytosolic protein) was saved for Western
blot analysis of IB, and the nuclear pellet was resuspended in the
high-salt buffer C (9) containing 20 mM HEPES (pH 7.6), 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 20 mM
-glycerophosphate, 10 mM
Na2MoO4,
50 µM
Na3VO4,
1 mM DTT, 1 mM PMSF, and the protease inhibitors cocktail described
previously. After being rotated at 4°C for up to 1 h, the nuclear
membranes were pelleted by microcentrifugation for 10 min and the clear supernatant (nuclear extract) was aliquoted and stored at
80°C. Protein concentration in the nuclear extract was
determined by the Bio-Rad protein assay (Bio-Rad Laboratories,
Hercules, CA).
Electrophoretic mobility shift assay.
Aliquots of nuclear extracts with equal amount of protein (2-10
µg) were mixed in 20 µl reactions with a buffer containing 10 mM
HEPES (pH 7.6), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10% (vol/vol) glycerol, and 3 µg poly[d(I-C)]. After aliquots were
equilibrated on ice for 5 min, binding reactions were started by the
addition of 20-60,000 counts/min (20 fmol) of
32P-labeled DNA probe and allowed
to proceed for 25-30 min at room temperature or up to 1 h on ice.
The oligonucleotide probe
5'-GCAGAGA containing a
B binding motif (underlined) was annealed to the complementary oligonucleotide with a 5'-G overhang and
end-labeled using Klenow DNA polymerase I. In mutated oligonucleotide,
the
B motif was changed (lowercase) to
. For cold
competition, a 60-300× molar excess of nonlabeled wild-type
or mutated
B oligonucleotide was added to the reaction together with
the probe. For supershift experiments, 1 µg of specific antibodies
against NF-
B proteins p65 (RelA), p50, or c-Rel (Santa Cruz
Biotechnology, Santa Cruz, CA) was added to the reaction 15 min before
the addition of the probe. Samples were electrophoresed at room
temperature in 0.5× TBE buffer (1× TBE 89 mM Tris base, 89 mM boric acid, 2 mM EDTA) on nondenaturing 4.5% polyacrylamide gel at
200 V. Gels were dried and directly analyzed in the PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) or exposed at
80°C to
Fuji RX film with intensifying screens. In this case the intensity of
bands on gel fluorograms was quantified using the image analysis system
AMBIS (Scanalytics, San Diego, CA).
Western blot analysis of IB.
Proteins in the cytosolic extract (see Preparation of nuclear
extracts) were separated by SDS-PAGE at 120 V using precast Tris-glycine gels and Mini-Cell gel apparatus (Novex, San Diego, CA). Separated proteins were electrophoretically
transferred to polyvinylidene difluoride membrane for 2 h at 30 V,
using a Novex blot module. Nonspecific binding was blocked by overnight
incubation of the membrane in 5% (wt/vol) nonfat dry milk in
Tris-buffered saline (TBS, pH 7.5). Blots were then incubated for 2 h
at room temperature with primary antibodies in antibody buffer
containing 1% (wt/vol) nonfat dry milk in TTBS (0.05% vol/vol Tween
20 in TBS), washed three times with TTBS, and finally incubated for 1 h
with a peroxidase-labeled secondary antibody in the antibody buffer.
Quantitation of mRNA levels for IL-6 and KC by RT-PCR. Total RNA was extracted from pancreatic tissue or isolated acinar cells using the method of Han et al. (23) or TRIzol reagent (GIBCO BRL). RNA quality was verified by better than 1:1 ethidium bromide staining of rRNA bands on denaturing agarose gel. Total RNA (5 µg) was reverse-transcribed according to the manufacturer's protocol (SuperScript II Preamplification System, GIBCO BRL) using oligo(dT) as a primer. cDNA prepared from 0.5 to 1 µg total RNA was subjected to PCR using gene-specific primers described below. Negative controls were performed by omitting the RT step from PCR amplification.
To quantitate changes in mRNA expression, we applied quantitative competitive (QC) PCR. For this analysis, a homologous external standard DNA (mimic) was created and coamplified with each of the cDNAs of interest, based on a modified procedure similar to that used previously (7, 29). Briefly, after the sense and antisense primers for the specific cDNA (target) were chosen, a "composite" primer was designed consisting of the sense primer spliced to a sequence 50-100 bp downstream in the cDNA of interest. Amplification of the target cDNA with the composite and antisense primers generated the mimic that therefore was 50-100 bp shorter than the expected gene-specific PCR product. After reamplification of the mimic with the sense and antisense primers, it was gel purified and quantified by spectrophotometry. Coamplification of a constant amount of target cDNA (derived from 0.5 µg of total RNA) with serial dilutions of its mimic allowed accurate quantitation of starting amount of target cDNA. Because the target cDNA and its mimic are coamplified with the same primer pair, share the same sequence, and have similar sizes, the difference in the efficiencies of their amplification is expected to be minimal. The amplified PCR products were all of expected size. They were separated on agarose gel stained with ethidium bromide, and the intensity of the bands was quantified by densitometry in GelBlot UVP (Synoptics, Upland, CA). The amount of specific mRNA of interest was determined from the competition curve derived by plotting the ratio of the densities of the target to mimic PCR products coamplified in the same tube vs. the concentration of the mimic (see Fig. 11A). The equivalence point, where this ratio equals 1.0, was estimated, and a correction was made for the difference in the target and mimic sizes. The reproducibility of our QC-PCR was tested by performing parallel series of PCR reactions on one and the same cDNA sample. For all of the PCR reactions the competition curve was well described by a straight line. In most cases the slope of this line was close to the theoretical value ofSerum amylase and lipase measurements. Serum amylase and lipase levels were determined by using a Hitachi 707 analyzer (Antech Diagnostics, Irvine, CA).
Acinar cell vacuolization. Pancreatic tissue was fixed immediately in 10% buffered formaldehyde. The tissue embedded in paraffin was sectioned and stained with hematoxylin and eosin. Values were obtained by counting the number of vacuoles per 100 acinar cells in an average of 50 fields at magnification ×40 (at least 1,000 acinar cells).
Determination of apoptotic acinar cells. Apoptotic acinar cells were determined as described previously (19, 45) on sections of pancreatic tissue stained with 8 mg/ml of Hoechst 33258. The stained sections were examined by fluorescence microscopy at excitation 380 nm and emission 510 nm. Scoring and classification of nuclei were performed based on the condensation state of chromatin. Acinar cells were considered apoptotic if their nuclei contained condensed and/or fragmented chromatin. A total of 5,000 to 10,000 cells were counted per slide.
Myeloperoxidase activity measurements. Myeloperoxidase (MPO) activity, a measure of neutrophil infiltration, was determined according to Bradley et al. (5). Briefly, the pancreatic tissue was homogenized in a solution containing 50 mM EDTA plus the cocktail of protease inhibitors (5 µg/ml each) pepstatin, aprotinin, leupeptin, chymostatin, and antipain, and 1 mM PMSF. The homogenate was centrifuged at 40,000 g for 15 min, the supernatant was discarded, and the pellet containing membrane-bound MPO was saved. MPO was solubilized by incubation with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0), 5 mM EDTA. Subsequently, the suspension was rehomogenized, sonicated, and centrifuged at 40,000 g for 15 min. The supernatant was collected, heated at 60°C for 2 h, and centrifuged at 10,000 g for 5 min. The supernatant containing MPO activity was saved. An aliquot of the supernatant was mixed with o-dianisidine and 0.005% hydrogen peroxide in 50 mM phosphate buffer (pH 6.0), and changes in absorbance at 460 nm were measured.
Measurements of active trypsin. Trypsin activity in pancreatic tissue homogenates was measured by a fluorometric assay according to the method of Kawabata et al. (28). Briefly, a 10-µl aliquot of the tissue homogenate was added to 2 ml of the assay buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM CaCl2, and 0.1 mg/ml BSA, in a stirred cuvette at 37°C. The reaction was started by adding the Boc-Gln-Ala-Arg-AMC substrate (28) and was followed for 6-10 min. The increase in fluorescence (excitation 380 nm, emission 440 nm) was linear during the observation. Trypsin activity in the homogenate was calculated using a standard curve for purified trypsin (Worthington, Freehold, NJ) obtained by the same procedure.
Statistical analysis of data. This was done using unpaired two-tailed Student's t-test.
Reagents.
Cerulein was obtained from Peninsula Laboratories (Belmont, CA). CCK
JMV-180
[Boc-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester] was from Research Plus (Bayonne, NJ). Boc-Gln-Ala-Arg-AMC was from
Bachem California (Torrance, CA). TRIzol Reagent and
Taq DNA polymerase were from GIBCO BRL
(Grand Island, NY). Exo(-) Klenow DNA polymerase I was from Stratagene
(La Jolla, CA). Poly(dI-dC) was from Boehringer Mannheim (Indianapolis,
IN). Polyclonal rabbit anti-mouse neutralizing TNF- antibody was
from Genzyme (Cambridge, MA). Antibodies against p65 (catalog number
sc-372 X), p50 (sc-114 X), and c-Rel (sc-71 X) NF-
B proteins, and
against I
B proteins were from Santa Cruz Biotechnology.
[
-32P]dCTP (3,000 Ci/mmol) was from Amersham (Arlington Heights, IL) or from Andotek
(Irvine, CA). All other reagents were from Sigma Chemical (St. Louis, MO).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NF-B is rapidly activated in rat cerulein
pancreatitis.
In the pancreas of normal and saline-infused rats, DNA binding activity
of NF-
B was virtually undetectable. Infusion of a supramaximal dose
(5 µg · kg
1 · h
1)
of cerulein caused a strong pancreatic NF-
B activation (Fig. 1). The specificity of NF-
B DNA binding
activity detected by our electrophoretic mobility shift assay (EMSA) in
the pancreas was demonstrated by cold competition experiments using
wild-type and mutated
B oligonucleotides (Fig. 1) and by supershift
experiments described below.
|
|
CCK JMV-180 does not activate NF-B in pancreas.
Infusion of a low, physiological dose of cerulein for 6 h did not evoke
NF-
B activation (Fig.
3A,
lane 8). To further prove that
NF-
B activation was associated with the development of the hormone-induced pancreatitis we made use of another derivative of CCK,
CCK JMV-180. This peptide, which binds to the same CCK-A receptor at
concentrations about 1,000 times higher than CCK, is considered to be
an agonist for the high-affinity state and antagonist for the
low-affinity state of the CCK-A receptor (26, 33, 46). In contrast to
CCK or cerulein, at high doses JMV-180 does not have an inhibitory
effect on pancreatic enzyme secretion (26, 33, 44, 46) nor does it
induce pancreatitis (44). Therefore, activation of a low-affinity state
of the CCK-A receptor is thought to mediate pancreatitis (44).
|
IB
and
I
B
display different kinetics of
degradation in cerulein pancreatitis.
Because NF-
B is activated via degradation of the inhibitory I
B
proteins we analyzed I
B
and I
B
in the cytosolic protein extracts from pancreatic tissue by Western blotting. Figure
4 shows that cerulein hyperstimulation
caused degradation of both I
B
and I
B
but with drastically
different kinetics. The I
B
was greatly decreased by the hormone
at the earliest time point studied (15 min), but it subsequently
recovered, and at 3 h cerulein infusion I
B
was at its normal
level in the control (Fig. 4). No degradation of I
B
was seen at 6 h cerulein infusion. In contrast, I
B
degradation developed more
slowly and was sustained for up to 6 h (Fig. 4). Thus the early phase
of cerulein-induced pancreatic NF-
B activation (Fig. 2) is due
mainly to I
B
degradation, whereas the second phase is mediated by
I
B
.
|
Cerulein directly activates NF-B in pancreatic
acinar cells.
To investigate whether acinar cells could be one source of
cerulein-induced pancreatic NF-
B activation, we asked if the hormone activates NF-
B in vitro. Dispersed acini, obtained from normal pancreas by standard collagenase digestion (41), displayed a certain
level of NF-
B DNA binding activity on isolation. However, incubation
of isolated acinar cells with cerulein caused a pronounced increase in
nuclear NF-
B compared with cells incubated without the hormone (Fig.
5A).
Acinar cells comprise >97% in our preparations of dispersed
pancreatic acini (19), and they do not contain any neutrophils or
macrophages, a potential source of activated NF-
B. Thus the hormone
directly activates NF-
B in acinar cells.
|
Subunit composition of pancreatic NF-B
complexes.
We used antibodies against different NF-
B proteins to characterize
subunit composition of pancreatic NF-
B complexes activated by
cerulein (Fig. 6). Both in tissue and in
isolated acinar cells stimulated with cerulein, two NF-
B-DNA bands
could be distinguished by EMSA, the lower (faster migrating) one being
usually more diffuse. Antibodies to NF-
B proteins p65 (RelA) and
p50, but not to c-Rel, either elicited a "supershifted" complex
or inhibited the DNA binding. The p50 antibody supershifted the lower
band and attenuated the upper one, whereas the antibody to p65
supershifted the upper band without changing the lower one, which is
seen more clearly on dispersed pancreatic acini EMSA (Fig.
6B). These data indicate that both
the classic p65/p50 heterodimer (slower migrating complex) and p50/p50
homodimer (the faster migrating complex) are formed on cerulein
hyperstimulation. The same types of NF-
B complexes are activated by
the hormone in vivo and in vitro. No complexes involving c-Rel could be
detected in pancreatic tissue (Fig.
6A) or dispersed acini (data not
shown).
|
TNF- involvement in NF-
B activation
in cerulein pancreatitis.
To provide an insight into the mechanism of pancreatic NF-
B
activation, we examined whether TNF-
had a role in this process. TNF-
is a potent activator of NF-
B (50, 55). Recently we showed
that pancreatic acinar cells are able to produce and respond to TNF-
and that neutralization of this cytokine with an antibody improved the
parameters of rat cerulein pancreatitis (19). Therefore, we tested if
the neutralizing anti-TNF-
antibody could prevent or inhibit NF-
B
activation induced by cerulein hyperstimulation. Immediately before
cerulein infusion, rats were given an intravenous bolus of the antibody
that was shown (19) to produce an improvement in pancreatitis (see
METHODS). This appeared to have no
effect on the early NF-
B activation caused by 30-min cerulein
infusion (Fig.
7A).
Moreover, the early phase of cerulein-induced NF-
B translocation was
not inhibited by pretreating the rat with one more such bolus of the
TNF-
antibody 1 h before hormone infusion (Fig.
7A, lane
3).
|
Blocking of NF-B activation improves parameters of
rat cerulein pancreatitis.
To study the role of NF-
B activation in the hormone-induced
pancreatitis, we used the antioxidant NAC to inhibit NF-
B. This compound is commonly used to block NF-
B activation (50, 55) and is
believed to act both via its direct reducing action and as glutathione
precursor (6, 11, 42, 47, 48). We applied NAC in various regimens, but
most of the data were obtained on rats that were given an intravenous
bolus of 200 mg/kg NAC 2 h before cerulein treatment followed by
continuous infusion of 25 mg · kg
1 · h
1
NAC for the period of the experiment. That is, a 6-h cerulein-infused rat received a total of 400 mg/kg (2.4 mmol/kg) NAC during 8 h. This
treatment almost completely abolished NF-
B activation caused by
cerulein hyperstimulation at both 30 min and 6 h (Fig.
8). For instance, NF-
B DNA binding
activity at the early 30-min peak decreased in NAC-treated animals as
much as 10-fold (n = 3) compared with
rats infused with cerulein only (Fig.
8A). In control (saline-infused) animals NAC treatment did not produce any changes in the measured parameters, e.g., serum amylase and lipase levels.
|
|
Pancreatic expression of NF-B-regulated genes for
cytokines IL-6 and KC is greatly activated in rat cerulein pancreatitis
and is inhibited by NAC.
To look into another functional aspect of NF-
B activation in
hormone-induced pancreatitis, we investigated the expression of
NF-
B-regulated genes for the acute phase cytokine IL-6 and the
chemokine KC (a murine analog of IL-8 and GRO-
). It has been shown
that patients with acute pancreatitis have elevated serum levels of
IL-6 and IL-8 that correlate with the severity of the disease (12, 16,
17, 25, 36). KC expression in experimental pancreatitis has not yet
been characterized, and the induction of IL-6 has not been quantified
(13, 36, 37).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results obtained in this study show that NF-B is rapidly and
strongly activated in rat cerulein pancreatitis. Within 15 min of
infusion of a supramaximal dose of the hormone, NF-
B DNA binding
activity in the pancreas increases several-fold, and it remains above
the basal level at 6 h cerulein hyperstimulation. Rapid kinetics is
typical for NF-
B activation. However, most of the data have been
obtained for cultured cells, and apart from lung and liver there are
few measurements of NF-
B in normal and injured tissues in the
development of a disease (3, 55). Except for abstract communications
(20-22, 35) and a recent observation of NF-
B activation in
biliary pancreatitis (10), this is the first detailed study of NF-
B
activation in the pancreas, in particular, of its role in
hormone-induced pancreatitis.
We observed a two-phase time course of the pancreatic NF-B
activation: an initial increase in DNA binding activity which peaked at
around 30 min and subsided by 1.5 h, and the second phase which was
most prominent at 3 h cerulein infusion. This kinetics can be explained
by the involvement of different species of inhibitory I
B proteins,
I
B
and I
B
, in the first and second phases of
hormone-induced NF-
B activation. We found that I
B
was rapidly
but transiently degraded in pancreatitis, and it completely returned to
normal levels at 3 and 6 h cerulein infusion. In contrast, degradation
of I
B
developed more slowly and was sustained up to 6 h. Thus the
early phase of pancreatic NF-
B activation is due mainly to I
B
degradation, whereas the second phase is mediated by I
B
.
The different behavior of IB
and I
B
is explained by
differences in transcriptional regulation of their genes by NF-
B. The gene encoding I
B
contains functional NF-
B binding sites and is transcriptionally activated by NF-
B, which leads to rapid resynthesis of I
B
and blockade of NF-
B nuclear translocation (1, 24, 27, 53, 55). Moreover, the resynthesized I
B
can diffuse
into the nucleus, bind there to NF-
B dimers, and dissociate them
from DNA sites (1, 56). In contrast to I
B
, no
B motifs have
been found in the gene for I
B
and its expression is not regulated
by NF-
B (1, 24, 27, 53, 55).
A biphasic NF-B nuclear translocation with a very similar time
course was reported recently in TNF-
-stimulated human hepatoblastoma cells (24). It was shown that the transient early (15 min to 1 h) phase
of NF-
B activation in these cells was due to a rapid I
B
resynthesis and that persistent I
B
proteolysis induced by TNF-
was necessary for the second (>2 h) phase.
Another factor contributing to the second wave of NF-B activation
could be inflammatory cell infiltration into the pancreas, which in the
rat model becomes pronounced after 2-3 h of cerulein hyperstimulation (14, 49). Inflammatory cells produce cytokines, such
as IL-1 and TNF-
, known to activate NF-
B. As shown by us and
others (13, 19, 36-38), TNF-
is upregulated in the pancreas during cerulein hyperstimulation. We showed that TNF-
induces NF-
B activation in isolated pancreatic acinar cells; we also found
that the acinar cell itself produced and released TNF-
(19). The
results obtained in the present study on the effects of TNF-
neutralization on NF-
B (Fig. 7) suggest the involvement of this
cytokine in NF-
B activation at 3 and 6 h of cerulein hyperstimulation but not at the early phase. The source of TNF-
could be both infiltrating inflammatory cells and acinar cells.
No NF-B binding activity was detected in response to a low,
physiological dose of cerulein or infusion of the CCK analog JMV-180
that does not induce pancreatitis. Furthermore, cerulein-induced NF-
B activation was abolished by coinfusion of JMV-180, a treatment that is known (44) to prevent pancreatitis. These results show that
pancreatic NF-
B activation caused by cerulein hyperstimulation is
associated with the development of the disease.
Several indirect lines of evidence in the present study implicate
acinar cells as one source of pancreatic NF-B activation in rat
cerulein pancreatitis: 1) the first,
main peak of NF-
B activation develops within 30 min of hormone
hyperstimulation, much faster than the infiltration of inflammatory
cells; 2) cerulein directly
activates NF-
B in isolated acinar cells through degradation of
I
B; and 3) the same types of
NF-
B complexes are activated by the hormone in pancreas and in
isolated acinar cells.
The isolation procedure caused a certain level of NF-B binding
activity in acinar cells, probably triggered by the disruption of
pancreatic extracellular matrix (18). However, incubation of acinar
cells with cerulein resulted in a marked NF-
B activation. Moreover,
we found that supramaximal concentrations of the hormone-induced degradation of both I
B
and I
B
in isolated acinar cells,
with kinetics similar to that observed in pancreatitis.
The results of supershift experiments indicate that at least two types
of NF-B complexes are activated in the pancreas of rats with
cerulein pancreatitis: p65/p50 and p50/p50 dimers. We have not detected
NF-
B complexes involving c-Rel. One may speculate that different
types of NF-
B complexes play distinct functional roles in
hormone-induced pancreatitis by selectively activating NF-
B target
genes with different binding sites (32, 50). On the other hand, p50
homodimers often behave as transcriptional inhibitors (32, 50, 55) and
in this way they may, for example, counteract an "excessive"
transactivation by p65/p50.
We used the antioxidant NAC to inhibit NF-B activation evoked by
cerulein. This compound is well tolerated and is commonly applied to
inhibit NF-
B in vivo (11, 47, 55). Both the early and late phases of
hormone-induced NF-
B activation were blocked by NAC. This resulted
in an improvement of all measures of rat cerulein pancreatitis,
suggesting that NF-
B activation is involved in the development of
the disease. Of importance, NAC significantly reduced pancreatic
trypsin activation, a key indicator of acinar cell injury. The only
parameter unchanged by NAC was the percentage of apoptotic acinar
cells, which can be affected by a number of factors involved in
pancreatitis (19, 45).
Besides NAC, with two more treatments we found a similar correlation
between inhibition of pancreatic NF-B and amelioration of rat
cerulein pancreatitis. One is TNF-
neutralization with an antibody
that both inhibited NF-
B activation (this study) and improved
cerulein-induced pancreatitis (19); another example is neutralization
of PAF by an antagonist, which inhibited both the development of
cerulein pancreatitis (45) and NF-
B activation (20).
NF-B plays a central role in regulating cytokine gene expression.
Using RT-PCR for quantitation of mRNA levels, we found that cerulein
hyperstimulation greatly induced mRNA expression of cytokine IL-6 and
chemokine KC. The latter is a murine analog of IL-8 and GRO-
, major
mediators of neutrophil activation and recruitment (2, 4). NF-
B
binding sites were found in the promoters of IL-8, KC, and IL-6, and
the expression of these genes in a number of cell lines was shown to be
regulated by NF-
B (4, 30, 40, 50, 55). Our results show that, in rat
cerulein pancreatitis, intrapancreatic mRNA levels for both IL-6 and KC increase up to 100-fold. This induction is inhibited by NAC, indicating the involvement of NF-
B in cytokine activation by the hormone.
Our quantitative analysis of IL-6 expression correlates well with the
data obtained by Northern blot and conventional RT-PCR (13, 36, 37).
mRNA expression of KC (or IL-8) in experimental pancreatitis has not
been characterized yet. In addition to IL-6 and KC, NF-B may also be
involved in the induction of other cytokines, in particular,
"first-line" cytokines IL-1 and TNF-
, which are upregulated in
cerulein-induced and other models of pancreatitis (36).
The observed upregulation of KC may play a role in activation and
recruitment of neutrophils into the pancreas. The neutrophils not only
mediate the inflammatory response but they also regulate acinar cell
death in the disease, as we recently found for rat cerulein
pancreatitis (45). The infiltrating inflammatory cells may contribute
to NF-B activation at later stages of pancreatitis.
Based on our results, several lines of indirect evidence suggest a
functional role for NF-B activation in cerulein pancreatitis: 1) hormone hyperstimulation causes a
rapid and strong NF-
B activation in the pancreas;
2) NF-
B activation is associated
with the development of the disease, as shown by experiments with
JMV-180; 3) cerulein directly
activates NF-
B in isolated acinar cells; both the kinetics of I
B
degradation and the subunit composition of activated NF-
B complexes
in vitro are similar to those observed in pancreatitis; 4) treatments with NAC, neutralizing
TNF-
antibody or PAF antagonist all both blunt pancreatitis and
inhibit NF-
B activation; and 5)
NF-
B-regulated genes for IL-6, KC, IL-1, and TNF-
are all induced
in the pancreas by hormone hyperstimulation. In this study we showed
that the expression of the first two is inhibited by NAC and
the last two have been shown to mediate cerulein pancreatitis (19,
36-39).
This evidence is of a correlative character, it only shows association
between NF-B inhibition and improvement of pancreatitis. More
specific, precise ways of inhibiting NF-
B, e.g., by using "decoy" NF-
B-binding oligonucleotides (34) or knockout animals (3, 55), are needed to prove the causative role of NF-
B activation
in the pathogenesis of pancreatitis.
NF-B activation was reported recently in a model of biliary
pancreatitis induced by duct ligation (10). Its inhibition by
amobarbital improved serum amylase levels, one of the parameters of
pancreatitis. Interestingly, from that study one can also infer a
correlation between NF-
B activation and the development of the
disease: biliary pancreatitis develops more slowly, and the NF-
B
activation is also delayed (Fig. 4 in Ref. 10) compared with cerulein pancreatitis.
In conclusion, based on the data obtained, we can hypothesize that
supramaximal doses of cerulein activate NF-B in acinar cells
resulting in upregulation of certain cytokines, like TNF-
, IL-6, and
KC, which mediate both acinar cell death (19) and inflammation (12,
36). The results suggest that NF-
B activation is an important early
event that may link the initial acinar cell injury to the inflammatory
and cell death responses, the hallmarks of acute pancreatitis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Hidekazu Tsukamoto for advice on EMSA, Sasa Periskic and David T. Kira for help with nuclear extract preparations, and Anne Taguchi for help in preparing this manuscript.
![]() |
FOOTNOTES |
---|
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: I. Gukovsky, West Los Angeles VA Medical Center, Bldg. 258, Rm. 340, 11301 Wilshire Blvd., Los Angeles, CA 90073.
Received 23 January 1998; accepted in final form 4 September 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arenzana-Seisdedos, F.,
J. Thompson,
M. S. Rodriguez,
F. Bachelerie,
D. Thomas,
and
R. T. Hay.
Inducible nuclear expression of newly synthesized IkBa negatively regulates DNA-binding and transcriptional activities of NF-kB.
Mol. Cell. Biol.
15:
2689-2696,
1995[Abstract].
2.
Baggiolini, M.,
B. Dewald,
and
B. Moser.
Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines.
Adv. Immunol.
55:
97-179,
1994[Medline].
3.
Barnes, P. J.,
and
M. Karin.
Nuclear factor-kB: a pivotal transcription factor in chronic inflammatory diseases.
N. Engl. J. Med.
336:
1066-1071,
1997
4.
Ben-Baruch, A.,
D. F. Michiel,
and
J. J. Oppenheim.
Signals and receptors involved in recruitment of inflammatory cells.
J. Biol. Chem.
270:
11703-11706,
1995
5.
Bradley, P. P.,
D. A. Priebat,
R. D. Christensen,
and
G. Rothstein.
Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.
J. Invest. Dermatol.
78:
206-209,
1982[Abstract].
6.
Brennan, P.,
and
L. A. O'Neill.
Effects of oxidants and antioxidants on nuclear factor kB activation in three different cell lines: evidence against a universal hypothesis involving oxygen radicals.
Biochim. Biophys. Acta
1260:
167-175,
1995[Medline].
7.
Celi, F. S.,
M. E. Zenilman,
and
A. R. Shuldiner.
A rapid and versatile method to synthesize internal standards for competitive PCR.
Nucleic Acids Res.
21:
1047,
1993[Medline].
8.
Dabrowski, A.,
A. Gabryelewicz,
and
L. Chyczewski.
The effect of platelet activating factor antagonist (BN 52021) on cerulein-induced acute pancreatitis with reference to oxygen radicals.
Int. J. Pancreatol.
8:
1-11,
1991[Medline].
9.
Dignam, J. D.,
R. M. Lebovitz,
and
R. G. Roeder.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:
1475-1489,
1983[Abstract].
10.
Dunn, J. A.,
C. Li,
T. Ha,
R. L. Kao,
and
W. Browder.
Therapeutic modification of nuclear factor kB binding activity and tumor necrosis factor-a gene expression during acute biliary pancreatitis.
Am. Surg.
63:
1036-1043,
1997[Medline].
11.
Flohe, L.,
R. Brigelius-Flohe,
C. Saliou,
M. G. Traber,
and
L. Packer.
Redox regulation of NF-kB activation.
Free Radic. Biol. Med.
22:
1115-1126,
1997[Medline].
12.
Formela, L. J.,
S. W. Galloway,
and
A. N. Kingsnorth.
Inflammatory mediators in acute pancreatitis.
Br. J. Surg.
82:
6-13,
1995[Medline].
13.
Fu, K.,
M. P. Sarras,
R. C. De Lisle,
and
G. K. Andrews.
Expression of oxidative stress-responsive genes and cytokine genes during caerulein-induced acute pancreatitis.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G696-G705,
1997
14.
Gorelick, F. S.,
G. Adler,
and
H. F. Kern.
Cerulein-induced pancreatitis.
In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by V. W. Go,
E. P. DiMagno,
J. D. Gardner,
E. Lebenthal,
H. A. Reber,
and G. A. Scheele. New York: Raven, 1993, p. 501-526.
15.
Grewal, H. P.,
A. M. el Din,
L. Gaber,
M. Kotb,
and
A. O. Gaber.
Amelioration of the physiologic and biochemical changes of acute pancreatitis using an anti-TNF-a polyclonal antibody.
Am. J. Surg.
167:
214-218,
1994[Medline].
16.
Gross, V.,
R. Andreesen,
H. G. Leser,
M. Ceska,
E. Liehl,
M. Lausen,
E. H. Farthmann,
and
J. Scholmerich.
Interleukin-8 and neutrophil activation in acute pancreatitis.
Eur. J. Clin. Invest.
22:
200-203,
1992[Medline].
17.
Gross, V.,
H. G. Leser,
A. Heinisch,
and
J. Schölmerich.
Inflammatory mediators and cytokinesnew aspects of the pathophysiology and assessment of severity of acute pancreatitis?
Hepatogastroenterology
40:
522-530,
1993[Medline].
18.
Gukovskaya, A. S.,
I. Gukovsky,
V. Zaninovic,
D. T. Kira,
and
S. J. Pandol.
Metalloproteinases regulate signal transduction pathways triggered by extracellular matrix proteins (Abstract).
Gastroenterology, Suppl.
114:
A464-A465,
1998.
19.
Gukovskaya, A. S.,
I. Gukovsky,
V. Zaninovic,
M. Song,
D. Sandoval,
S. Gukovsky,
and
S. J. Pandol.
Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-a. Role in regulating cell death and pancreatitis.
J. Clin. Invest.
100:
1853-1862,
1997
20.
Gukovsky, I.,
T. A. Blinman,
V. Zaninovic,
Y. Jung,
A. S. Gukovskaya,
and
S. J. Pandol.
Role of transcription factor NF-kB in mediating hormone-induced pancreatitis (Abstract).
Gastroenterology, Suppl.
114:
A465,
1998.
21.
Gukovsky, I.,
A. S. Gukovskaya,
and
S. J. Pandol.
Cerulein activates NF-kB and AP-1 in isolated pancreatic acinar cells (Abstract).
Gastroenterology, Suppl.
114:
A465,
1998.
22.
Gukovsky, I.,
A. S. Gukovskaya,
V. Zaninovic,
S. Periskic,
H. Tsukamoto,
and
S. J. Pandol.
NF-kB activation is an early marker of pancreatic acinar cell injury in rat cerulein pancreatitis (Abstract).
Gastroenterology, Suppl.
112:
A446,
1997.
23.
Han, J. H.,
C. Stratowa,
and
W. J. Rutter.
Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning.
Biochemistry
26:
1617-1625,
1987[Medline].
24.
Han, Y.,
and
A. R. Brasier.
Mechanism for biphasic Rel A/NF-kB1 nuclear translocation in tumor necrosis factor a-stimulated hepatocytes.
J. Biol. Chem.
272:
9825-9832,
1997
25.
Heath, D. I.,
A. Cruickshank,
M. Gudgeon,
A. Jehanli,
A. Shenkin,
and
C. W. Imrie.
Role of interleukin-6 in mediating the acute phase protein response and potential as an early means of severity assessment in acute pancreatitis.
Gut
34:
41-45,
1993[Abstract].
26.
Jensen, R. T.,
S. A. Wank,
W. H. Rowley,
S. Sato,
and
J. D. Gardner.
Interaction of CCK with pancreatic acinar cells.
Trends Pharmacol. Sci.
10:
418-423,
1989[Medline].
27.
Johnson, D. R.,
I. Douglas,
A. Jahnke,
S. Ghosh,
and
J. S. Pober.
A sustained reduction in IkB-b may contribute to persistent NF-kB activation in human endothelial cells.
J. Biol. Chem.
271:
16317-16322,
1996
28.
Kawabata, S.,
T. Miura,
T. Morita,
H. Kato,
K. Fujikawa,
S. Iwanaga,
K. Takada,
T. Kimura,
and
S. Sakakibara.
Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin.
Eur. J. Biochem.
172:
17-25,
1988[Abstract].
29.
Koehler, T.,
D. Labner,
A.-K. Rost,
B. Thamm,
B. Pustowoit,
and
H. Remke.
Quantitation of mRNA by Polymerase Chain Reaction. Berlin: Springer, 1995.
30.
Kunsch, C.,
R. K. Lang,
C. A. Rosen,
and
M. F. Shannon.
Synergistic transcriptional activation of the IL-8 gene by NF-kB p65 (RelA) and NF-IL-6.
J. Immunol.
153:
153-164,
1994
31.
Lerch, M. M.,
and
G. Adler.
Experimental animal models of acute pancreatitis.
Int. J. Pancreatol.
15:
159-170,
1994[Medline].
32.
Liou, H. C.,
and
D. Baltimore.
Regulation of the NF-kB/Rel transcription factor and IkB inhibitor system.
Curr. Opin. Cell Biol.
5:
477-487,
1993[Medline].
33.
Matozaki, T.,
J. Martinez,
and
J. A. Williams.
A new CCK analogue differentiates two functionally distinct CCK receptors in rat and mouse pancreatic acini.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G594-G600,
1989
34.
Morishita, R.,
T. Sugimoto,
M. Aoki,
I. Kida,
N. Tomita,
A. Moriguchi,
K. Maeda,
Y. Sawa,
Y. Kaneda,
J. Higaki,
and
T. Ogihara.
In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction.
Nat. Med.
3:
894-899,
1997[Medline].
35.
Neuschwander-Tetri, B. A.,
and
L. D. Wells.
Early activation of pancreatic NF-kB in cerulein-induced acute pancreatitis in the mouse (Abstract).
Gastroenterology, Suppl.
110:
A421,
1996.
36.
Norman, J.
The role of cytokines in the pathogenesis of acute pancreatitis.
Am. J. Surg.
175:
76-83,
1998[Medline].
37.
Norman, J. G.,
G. W. Fink,
W. Denham,
J. Yang,
G. Carter,
C. Sexton,
J. Falkner,
W. R. Gower,
and
M. G. Franz.
Tissue-specific cytokine production during experimental acute pancreatitis. A probable mechanism for distant organ dysfunction.
Dig. Dis. Sci.
42:
1783-1788,
1997[Medline].
38.
Norman, J. G.,
G. W. Fink,
and
M. G. Franz.
Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression.
Arch. Surg.
130:
966-970,
1995[Abstract].
39.
Norman, J. G.,
M. G. Franz,
G. S. Fink,
J. Messina,
P. J. Fabri,
W. R. Gower,
and
L. C. Carey.
Decreased mortality of severe acute pancreatitis after proximal cytokine blockade.
Ann. Surg.
221:
625-631,
1995[Medline].
40.
Ohmori, Y.,
S. Fukumoto,
and
T. A. Hamilton.
Two structurally distinct kB sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages.
J. Immunol.
155:
3593-3600,
1995[Abstract].
41.
Pandol, S. J.,
R. T. Jensen,
and
J. D. Gardner.
Mechanism of [Tyr4]bombesin-induced desensitization in dispersed acini from guinea pig pancreas.
J. Biol. Chem.
257:
12024-12029,
1982
42.
Pinkus, R.,
L. M. Weiner,
and
V. Daniel.
Role of oxidants and antioxidants in the induction of AP-1, NF-kB, and glutathione S-transferase gene expression.
J. Biol. Chem.
271:
13422-13429,
1996
43.
Raeymaekers, L.
Quantitative PCR: theoretical considerations with practical implications.
Anal. Biochem.
214:
582-585,
1993[Medline].
44.
Saluja, A. K.,
M. Saluja,
H. Printz,
A. Zavertnik,
A. Sengupta,
and
M. L. Steer.
Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion.
Proc. Natl. Acad. Sci. USA
86:
8968-8971,
1989[Abstract].
45.
Sandoval, D.,
A. Gukovskaya,
P. Reavey,
S. Gukovsky,
A. Sisk,
P. Braquet,
S. J. Pandol,
and
S. Poucell-Hatton.
The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis.
Gastroenterology
111:
1081-1091,
1996[Medline].
46.
Sato, S.,
H. A. Stark,
J. Martinez,
M. A. Beaven,
R. T. Jensen,
and
J. D. Gardner.
Receptor occupation, calcium mobilization, and amylase release in pancreatic acini: effect of CCK-JMV-180.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G202-G209,
1989
47.
Schreck, R.,
and
P. A. Baeuerle.
Assessing oxygen radicals as mediators in activation of inducible eukaryotic transcription factor NF-kappa B.
Methods Enzymol.
234:
151-163,
1994[Medline].
48.
Schreck, R.,
P. Rieber,
and
P. A. Baeuerle.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1.
EMBO J.
10:
2247-2258,
1991[Abstract].
49.
Shimizu, I.,
S. Wada,
T. Okahisa,
M. Kamamura,
M. Yano,
T. Kodaira,
T. Nishino,
K. Shima,
and
S. Ito.
Radioimmunoreactive plasma bradykinin levels and histological changes during the course of cerulein-induced pancreatitis in rats.
Pancreas
8:
220-225,
1993[Medline].
50.
Siebenlist, U.,
G. Franzoso,
and
K. Brown.
Structure, regulation and function of NF-kappa B.
Annu. Rev. Cell Biol.
10:
405-455,
1994.
51.
Steer, M. L.,
and
A. K. Saluja.
Experimental acute pancreatitis: studies of the early events that lead to cell injury.
In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by V. W. Go,
E. P. DiMagno,
J. D. Gardner,
E. Lebenthal,
H. A. Reber,
and G. A. Scheele. New York: Raven, 1993, p. 489-500.
52.
Steinberg, W.,
and
S. Tenner.
Acute pancreatitis.
N. Engl. J. Med.
330:
1198-1210,
1994
53.
Thompson, J. E.,
R. J. Phillips,
H. Erdjument-Bromage,
P. Tempst,
and
S. Ghosh.
IkB-b regulates the persistent response in a biphasic activation of NF-kB.
Cell
80:
573-582,
1995[Medline].
54.
Willemer, S., H. P. Elsasser, and G. Adler.
Hormone-induced pancreatitis. Eur. Surg.
Res. 24, Suppl. 1:
29-39, 1992.
55.
Wulczyn, F. G.,
D. Krappmann,
and
C. Scheidereit.
The NF-kB/Rel and IkB gene families: mediators of immune response and inflammation.
J. Mol. Med.
74:
749-769,
1996[Medline].
56.
Zabel, U.,
and
P. A. Baeuerle.
Purified human IkB can rapidly dissociate the complex of the NF-kB transcription factor with its cognate DNA.
Cell
61:
255-265,
1990[Medline].
57.
Zhou, W.,
B. A. Levine,
and
M. S. Olson.
Platelet-activating factor: a mediator of pancreatic inflammation during cerulein hyperstimulation.
Am. J. Pathol.
142:
1504-1512,
1993[Abstract].