Department of Medicine, Veterans Affairs Greater Los Angeles Healthcare System and the University of California, Los Angeles, Los Angeles, California 90073
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcription factor nuclear
factor-B (NF-
B) is activated in cerulein pancreatitis and
mediates cytokine expression. The role of transcription factor
activation in other models of pancreatitis has not been established.
Here we report upregulation of NF-
B and inflammatory molecules, and
their correlation with local pancreatic injury, in a model of severe
pancreatitis. Rats received intraductal infusion of taurocholate or
saline, and the pancreatic head and tail were analyzed separately.
NF-
B and activator protein-1 (AP-1) activation were assessed by gel
shift assay, and mRNA expression of interleukin-6, tumor necrosis
factor-
, KC, monocyte chemoattractant protein-1, and inducible
nitric oxide synthase was assessed by semiquantitative RT-PCR.
Morphological damage and trypsin activation were much greater in the
pancreatic head than tail, in parallel with a stronger activation of
NF-
B and cytokine mRNA. Saline infusion mildly affected these
parameters. AP-1 was strongly activated in both pancreatic segments
after either taurocholate or saline infusion. NF-
B inhibition with
N-acetylcysteine ameliorated the local inflammatory
response. Correlation between localized NF-
B activation, cytokine
upregulation, and tissue damage suggests a key role for NF-
B in the
development of the inflammatory response of acute pancreatitis.
acute pancreatitis; nuclear factor-B; activator protein-1; cytokines; chemokines; inducible nitric oxide synthase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ALTHOUGH THE MECHANISM OF acute pancreatitis has not been established, there is emerging evidence that upregulation of inflammatory mediators, such as cytokines, chemokines, adhesion molecules, and inducible nitric oxide synthase (iNOS), is central to the process (for recent reviews, see Refs. 6, 36, and 43). Upregulation of these molecules has been shown in human (5, 20, 24, 28) and experimental pancreatitis (11, 12, 16, 17, 38, 39, 41, 48, 52), and their role in the disease has been demonstrated by using pharmacological inhibitors or antagonists (17, 21, 35, 37, 41, 47), neutralizing antibodies (16, 38, 48, 52), and genetic approaches (7, 8, 12).
The cellular mechanism of regulation of these inflammatory molecules
involves activation of transcription factors such as nuclear factor
(NF)-B (3, 14, 51) and activator protein (AP)-1
(50). Activation of NF-
B in the pancreas has recently been demonstrated in rat cerulein pancreatitis (17, 44).
Inhibition of NF-
B activation resulted in a decrease in the
expression of cytokines KC [rat analog of interleukin (IL)-8/Gro-
]
and IL-6 (17).
NF-B and AP-1 represent two early response transcriptional complexes
essential for the gene expression of inflammatory molecules (3,
14, 15, 50). NF-
B exists as a complex of homo- or heterodimers composed of members of the Rel family of proteins (3, 14). In most resting cells, NF-
B is sequestered
within the cytoplasm in an inactive form. After activation, NF-
B
complexes translocate into the nucleus and activate transcription from
target genes. AP-1 represents a homo- or heterodimer complex composed of Jun, Fos, or activating transcription factor subunits (22, 49). These components normally reside in the nucleus and after stimulation bind to phorbol ester- or cAMP-responsive sites on DNA,
inducing gene expression.
The involvement of the inflammatory molecules and their regulation has
mostly been investigated in rat cerulein-induced pancreatitis, a model
with mild severity (1, 26). To establish a universal role
for these mechanisms in the development of pancreatitis, we designed
the present set of experiments in a model of pancreatitis induced in
rats by the taurocholate biliopancreatic duct infusion. This model is
associated with severe pancreatic necrosis and a high mortality
(2, 27). We analyzed early molecular events, namely
activation of inflammatory molecules [IL-6, tumor necrosis factor
(TNF)-, KC, monocyte chemoattractant protein (MCP)-1, and iNOS] and
the transcription factors involved in their regulation (NF-
B and
AP-1).
In the taurocholate model, pancreatic damage is mainly localized to the head of the pancreas, whereas in cerulein-induced pancreatitis and other models the damage is evenly distributed throughout the organ. Taking advantage of this characteristic of taurocholate-induced pancreatitis, we studied the early molecular events separately in the pancreatic head and tail and correlated them with the local morphological and biochemical changes (e.g., neutrophil infiltration and trypsin activation). Furthermore, because several factors (chemical and mechanical effect, oxidative stress, ischemia-reperfusion; see Refs. 2, 25, 31, 45) are involved in the mechanism of this model, we investigated the pancreatic response to saline intraductal infusion, in which the trigger is restricted to increased hydrostatic pressure. Characterization of the injury induced by saline infusion may have a direct clinical implication for understanding the acute pancreatitis that some patients develop as a complication of the endoscopic retrograde cholangiopancreatography (18, 29).
We found localized activation of NF-B (but not AP-1) and localized
upregulation of inflammatory molecules in the pancreatic head. These
molecular changes correlated with the severity of morphological
changes, including inflammatory cell infiltration, and with trypsin
activation. Moreover, pharmacological blockade of NF-
B activation
resulted in attenuation of the local inflammatory response. Saline
infusion caused, although to a lesser degree, characteristic
morphological changes of pancreatitis and NF-
B and AP-1 activation
and upregulation of inflammatory molecules. The results indicate that
taurocholate infusion causes a localized activation of NF-
B that, in
turn, upregulates the expression of inflammatory molecules. These
signals may be important for the infiltration of inflammatory
cells and localized parenchymal damage in pancreatitis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Ketamine hydrochloride was from Fort Dodge Laboratories (Fort Dodge, IA), and xylazine was from Miles (Shawnee Mission, KS); Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin (AMC) was from Bachem (Torrance, CA); PE-50 tubing was from Becton-Dickinson (Sparks, MD); trypsin was from Worthington (Freehold, NJ); MES was from ICN Biomedicals (Aurora, OH); Tissue-Tek optimum cutting temperature (OCT) compound was from Sakura Finetek (Torrance, CA); polyclonal rabbit anti-rat neutrophil antibody was from Accurate Chemical and Science (Westbury, NY); antibodies to NF-Experimental Procedure
Care and handling of the animals were approved by the Animal Research Committee of the Veterans Affairs Greater Los Angeles Healthcare System, in accordance with National Institutes of Health guidelines. Male Sprague-Dawley rats (Harlan, Madison, WI), weighing 300-340 g, were randomized into different groups and fasted overnight before the experiment. Acute taurocholate pancreatitis was induced according to Aho et al. (2). Under a combination of xylazine (10 mg/kg) and ketamine (100 mg/kg) anesthesia, a midline laparotomy was performed and a PE-50 catheter (inside diameter 0.58 mm, outside diameter 0.96 mm) was inserted in the pancreatic duct through a puncture of the duodenum. The biliopancreatic duct was occluded transiently by a silk ligature at the liver hilus to prevent regurgitation of the infusate into the liver. Sodium taurocholate solution (0.4 ml of 4%; TC group) or the same volume of saline (saline group) was retrogradely infused in the pancreatic duct at a flow rate of 0.07 ml/min by a microinfusion pump. At the completion of the infusion, the catheter and hilar ligature were removed and the abdomen was closed with suture. Intact rats served as a control (control group). To inhibit NF-At 1 or 6 h after taurocholate infusion, animals were killed by
CO2 asphyxiation and blood was obtained in heparinized
tubes for amylase and lipase determinations. Portions of the pancreas were immediately removed, trimmed of fat, rinsed in ice-cold PBS, snap
frozen in liquid nitrogen, and stored at 80°C until processing for
active trypsin measurements, nuclear protein extraction, and RNA
isolation. Portions of the gland were also saved for morphological examination. Specimens of the head and tail segments of the pancreas were collected and analyzed separately. For histological examination, fresh pancreatic tissue was fixed in 10% zinc-buffered formalin, embedded in paraffin, and cut into 3- to 5-µm-thick sections. For
immunostaining, 2- to 4-mm pieces of pancreas were fixed in 4%
paraformaldehyde (pH 7.4) at 4°C for 2 h and dehydrated in an
increasing gradient of sucrose solutions (0.5, 1, and 2.3 M) at 4°C.
Next, the tissue was frozen in Tissue-Tek OCT and stored at
80°C.
Immunohistochemistry
For immunohistochemical detection of neutrophils, 8-µm-thick cryosections were mounted on poly-L-lysine-coated glass slides. After being washed with PBS for 5 min and then with PBS containing 0.05 M glycine (PBS/glycine) for 10 min, sections were incubated in a blocking medium containing PBS/glycine, 1% (vol/vol) goat serum, 1% (wt/vol) BSA, and 1% (wt/vol) gelatin. After 20 min, tissue sections were transferred to working medium (PBS/glycine, 0.1% goat serum, 0.1% BSA, and 0.1% gelatin) for 10 min followed by a 2-h incubation with rabbit anti-rat polymorphonuclear leukocyte antibody conjugated with FITC at 1:125 dilution. The sections were then washed three times with PBS and air dried. Anti-fading mixture (4% propyl gallate and 90% glycerol in PBS, pH 7.8) was added, and the FITC-stained neutrophils were imaged by fluorescence microscopy using a Nikon Diaphot microscope.For p65 immunolocalization, paraffin sections and the ABC Peroxidase Staining Kit were used. Endogenous peroxidase activity was blocked by tissue incubation in 0.3% H2O2 in methanol for 30 min. Slides were washed with PBS for 5 min and then with PBS containing 0.05 M glycine for 10 min. Sections were blocked in the blocking medium for 20 min, washed for 10 min in the working medium, and incubated overnight at 4°C with anti-p65 antibody at 1:50 dilution. Slides were then washed with PBS three times for 10 min and incubated for 2 h at room temperature with biotin-conjugated secondary antibody diluted in the working buffer (1:200). Sections were washed with PBS/glycine buffer and incubated with ABC peroxidase reagent for 30 min, then washed with PBS/glycine buffer three times for 10 min, incubated with diaminobenzidine substrate and H2O2, washed in PBS/glycine buffer three times for 10 min, and examined by light microscopy. The specificity of p65 staining was confirmed by the use of secondary reagents, omitting the primary antibody.
Values were obtained by counting the number of neutrophils (or the number of acinar cell nuclei immunoreactive for p65) per 100 acinar cells. An average of 100 fields was counted for neutrophils, and an average of 20 fields was counted for p65 immunolocalization from four to six rats for each condition.
Assays
Measurement of active trypsin. Trypsin activity in pancreatic tissue homogenates was measured by a fluorimetric assay according to the method of Kawabata et al. (23). Briefly, the tissue was homogenized on ice in 3 vol of a buffer containing 5 mM MES (pH 6.5), 1 mM MgSO4, and 250 mM sucrose using a glass-Teflon homogenizer. A 25-µl aliquot of the 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-Glu-Ala-Arg-AMC substrate and was followed for 5 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 obtained by the same procedure.
Preparation of nuclear extracts.
Nuclear protein extracts from pancreatic tissue were prepared for gel
shift assay as described earlier (17, 39). Briefly, 100-200 mg of frozen tissue were powdered in a mortar in liquid nitrogen and lysed in 1 ml of a low-salt buffer (17) with
20 strokes in a glass Dounce homogenizer. Before use, the lysis buffer was supplemented with phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) to a final concentration of 1 mM each and with the
protease-inhibitor cocktail containing 5 µg/ml each of pepstatin,
leupeptin, chymostatin, antipain, and aprotinin. After 20 min, 10%
Igepal CA-630 was added to a final concentration of 0.3% (vol/vol),
and then nuclei were collected by 30 s microcentrifugation. Nuclear protein was extracted from pelleted nuclei in a high-salt buffer (17) supplemented with 1 mM PMSF, 1 mM DTT, and the
protease inhibitor cocktail described previously for up to 1 h at
4°C. Nuclear membranes were pelleted by microcentrifugation for 10 min, and the clear supernatant (nuclear extract) was separated into
aliquots and stored at 80°C. The protein content of the nuclear
extract was determined using the Bio-Rad protein assay.
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA) experiments were performed
using double-stranded oligonucleotides comprising the consensus
sequences (underlined) for NF-B
(5'-GCAGAGGGGACTTTCCGAGA) and AP-1
(5'-GGCTTGATGAGTCAGCCGGAA). The binding site for AP-1 comprised the phorbol ester-responsive element. Oligonucleotides were
end labeled with [32P]dCTP using Klenow DNA polymerase I. In the mutated NF-
B oligonucleotide, the
B motif was changed to
GGccACTaaCC. Nuclear proteins (7-10 µg) were incubated with the
32P-labeled oligonucleotide probe under binding conditions
[10 mM HEPES (pH 7.8), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10%
(vol/vol) glycerol, and 3 µg poly(dI-dC)] for 20 min at room
temperature in a final volume of 20 µl. For cold competition, a 100×
molar excess of unlabeled wild-type or mutated oligonucleotides was added with the probe. For supershift experiments, 2-4 µg of specific antibodies against NF-
B proteins p65 (Rel A), p50, p52, and c-Rel or
against AP-1 proteins c-Fos, c-Jun, Jun-B, Jun-D, and Fra-2 were
incubated with the binding reaction mixture for 40 min at room
temperature before addition of the probe. After binding, protein-DNA
complexes were electrophoresed on a native 4.5% polyacrylamide gel at
200 volts using 0.5× TBE buffer (1×x TBE: 89 mM Tris base, 89 mM
boric acid, and 2 mM EDTA). After being dried, the gels were quantified
in the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Semiquantitative RT-PCR analysis of mRNA expression.
The procedure for semiquantitative RT-PCR was essentially as described
previously (17, 39). Total RNA was extracted from pancreatic tissue using the TRIzol reagent. RNA quality was verified by
ethidium bromide staining of ribosomal RNA bands on denaturing agarose
gel. Total RNA (5 µg) was reverse-transcribed using oligo(dT) as a
primer (SuperScript II Preamplification System). cDNA obtained from 0.5 µg total RNA was subjected to PCR using rat gene-specific primers for
IL-6, TNF-, KC, MCP-1, and iNOS. The primer sequences were described
previously (17, 39). Amplification was for a different
number of cycles (between 22 and 32) to yield visible products within
the linear amplification range. For a given target sequence, the same
cycle number was applied for cDNAs from all animals studied. Negative
controls were performed by omitting the RT step or the cDNA template
from PCR amplification. The identity of the RT-PCR products was
confirmed by direct sequencing. They were separated on an agarose gel
containing ethidium bromide, photographed, and quantified using an
AMBIS image analysis system (Scanalytics, San Diego, CA). Background
correction in the densitometry was performed using an area immediately
adjacent to the RT-PCR product band. mRNA levels were normalized to the
density of the RT-PCR product for acidic ribosomal phosphoprotein P0
(ARP), used as a housekeeping gene, in the same sample, as we described
previously (17, 39).
Statistical Analysis
Data are presented as means ± SE of the values. Statistical differences between values from two groups were determined by the unpaired Student's t-test. For normally distributed parameters, differences between more than two groups were determined by one-way ANOVA, and multiple comparisons were performed by using Newman-Keuls post hoc analysis. For parameters without normal distribution, differences between more than two groups were determined by the Kruskal-Wallis test and Dunn's post hoc analysis. A P value <0.05 was considered statistically significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Macroscopic and Histological Examination of the Pancreas
Rats that received intraductal infusion of saline showed slight pancreatic edema, and light microscopy revealed a mild degree of interstitial edema. By contrast, after 1 or 6 h of taurocholate infusion, all of the animals had severe necrohemorrhagic pancreatitis, observed grossly and by light microscopy, that mostly affected the head segment of the pancreas. The pancreatic tail had normal histology in most of the animals.Parameters of Pancreatitis: Serum Amylase and Lipase, Pancreatic Neutrophil Infiltration, and Trypsin Activation
Figure 1 shows the levels of serum amylase and lipase and the infiltration of neutrophils in pancreatic tissue in untreated rats and in those at 1 or 6 h after receiving intraductal infusion of either saline or taurocholate. Animals infused with taurocholate showed a time-dependent increase of serum amylase and lipase that was already significant after 1 h of the infusion. Intraductal saline infusion induced a smaller increase in serum amylase and lipase that was significantly higher compared with the control group.
|
The number of infiltrating neutrophils in the pancreatic head of taurocholate-infused rats followed a similar time-dependent pattern as the amylase and lipase levels. The number of neutrophils in the pancreatic head increased also in the 6-h saline group, but to a lesser extent. In control pancreata, no inflammatory infiltration was seen (Fig. 1C).
Trypsin activation was also evaluated separately in the pancreatic head
and tail (Fig. 2). In the head of the
pancreas, trypsin activity increased at 1 h and then further at
6 h after taurocholate infusion. The saline treatment induced a
smaller increase that was statistically significant in the 6-h saline
group compared with control and 1-h saline groups. Trypsin activity in
the pancreatic tail after taurocholate infusion showed a much smaller
increase than in the head (P < 0.05) and was not
statistically different from the control group.
|
Activation of Pancreatic NF-B in Taurocholate-Induced
Pancreatitis
|
|
Composition of NF-B Complexes
To further analyze cellular localization of the activated NF-B, we
performed immunostaining in pancreatic tissue with a specific antibody
to p65. NF-
B is kept in inactive form in the cytoplasm and
translocates to the nucleus upon activation. Thus immunostained nuclei
indicate the presence of the activated NF-
B. In accordance with EMSA
data, p65 immunoreactivity in the cell nuclei was not observed in
control pancreas (Fig. 5A). In
contrast, pancreatic tissue from the 6-h taurocholate group, in which a
strong NF-
B activation was detected by EMSA, displayed immunostained
nuclei, confirming the activation of NF-
B (Fig. 5B). In
both control and pancreatitis tissue, no staining was observed without
primary antibody (data not shown). Most of immunoreactivity was
confined to the acinar cell population, and ~10% of the acinar cell
nuclei stained for p65 (Fig. 5B). A small portion of
infiltrated neutrophils also showed nuclear p65 localization (Fig.
5C).
|
Activation of Pancreatic AP-1 Complexes in Taurocholate-Induced Pancreatitis
In the pancreas of normal rats, there was essentially no AP-1 DNA binding activity. As shown in Fig. 6, AP-1 was strongly activated in pancreata from rats at 1 and 6 h after infusion of either saline or taurocholate. Thus, compared with NF-
|
The specificity of AP-1 binding was confirmed in cold competition
experiments (Fig. 7A) using
unlabeled wild-type or unrelated (Oct-1) oligonucleotide.
|
AP-1 is a dimeric transcription factor that consists of homodimers of the Jun family proteins (c-Jun, Jun-B, and Jun-D) or Jun/Fos heterodimers. Members of the Fos family (c-Fos, Fos-B, Fra-1, and Fra-2) can associate with different Jun proteins to form the heterodimeric AP-1 complexes. The composition of AP-1 complexes in pancreata of taurocholate-infused animals was determined by supershift analysis using antibodies against Jun or Fos proteins (Fig. 7B). A supershifted band was detected with c-Fos, Jun-B, and Fra-2 antibodies. The addition of Jun-B antibody also caused a significant decrease of the specific AP-1 band (Fig. 7B). c-Jun, Jun-D, and Fos-B did not demonstrate a clear supershift or intensity decrease of the AP-1 band. Thus the results obtained indicate the presence of c-Fos, Jun-B, and Fra-2 in AP-1 complex(es) activated in the pancreas of taurocholate-infused rats.
Cytokine, Chemokine, and iNOS mRNA Expression in Pancreatic Tissue
To determine whether there is upregulation of gene expression for inflammatory molecules in the pancreas, mRNA levels of the cytokines IL-6 and TNF-
|
Regional Activation of NF-B, AP-1, and Inflammatory Molecule
Gene Expression in the Pancreas
|
Similarly, the mRNA expression for IL-6, MCP-1, iNOS (Fig. 9C), and KC (data not shown) also displayed a localized response, with strong upregulation in the head of the pancreas contrasting the absent or weak expression in the corresponding tail segment.
Abrogation of the Local Inflammatory Response in
Taurocholate-Induced Pancreatitis by NF-B Inhibition
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There is emerging consensus that inflammatory mediators
(cytokines, in particular) play a central role in the development of
experimental acute pancreatitis (6, 36, 43). Recently, NF-B has been identified as an important regulator that controls the
expression of many of those inflammatory mediators in the pancreas
(9, 17, 19, 39, 44, 52). These molecular events have been
studied in more detail in the rat cerulein model of pancreatitis.
Administration of the CCK analog cerulein in rats causes a very
reproducible but mild form of pancreatitis limited to changes in the
pancreas, without major systemic complications or lethality (1,
26). By contrast, taurocholate infusion into the rat
biliopancreatic duct is associated with severe local pancreatic damage
(edema, necrosis, hemorrhage, and abundant leukocyte infiltration),
major systemic complications, and high mortality (2). This
model closely reproduces the necrohemorrhagic pancreatitis in humans
(27). In the present study, we chose the
taurocholate-induced pancreatitis model to investigate whether the
above-described molecular events are common between different
experimental models and, hence, represent a common pathway in the early
phase of the disease. In addition to NF-
B, we also studied AP-1,
another important transcription factor in the early inflammatory
response (15, 50). Because in the taurocholate model the
damage is mostly localized to the pancreatic head, we asked whether
activation of transcription factors and inflammatory molecules
correlated with parameters of pancreatitis. Finally, we analyzed
responses to the mechanical effect of saline intraductal infusion.
Morphologically, taurocholate infusion caused severe pancreatic damage
in the head of the pancreas, evident at both 1 and 6 h after the
infusion. This was accompanied by a progressive increase in serum
amylase and lipase, pancreatic neutrophil infiltration, and trypsin
activation in the head segment of the gland. After a parallel time
course, NF-B was rapidly activated in the pancreatic head, with
increasing activation at 6 h after taurocholate infusion. These
observations extend previous findings of early pancreatic NF-
B
activation in the rat cerulein model (17, 44) and in pancreatitis induced by occlusion of the pancreatic duct
(9). In those studies, NF-
B activation was detected
within the first 15 min of cerulein induction (17, 44) and
1 h after pancreatic duct occlusion (9). NF-
B
activation has also been reported in peritoneal and alveolar
macrophages from rats with taurocholate-induced pancreatitis
(42).
With the supershift assay, we showed that p65 (Rel A) and p50 subunits
are present in the activated NF-B complexes in taurocholate pancreatitis. We immunolocalized the translocated p65 subunit to the
acinar cell nuclei. p65 translocation into acinar cell nuclei has been
also observed in the cerulein model (44). Although neutrophil infiltration into the pancreas was abundant 6 h after taurocholate infusion, the immunostaining revealed that the main source
of nuclear p65 was the acinar cells. This suggests that NF-
B
activation in the acinar cells represents a primary event in the early
phase of acute pancreatitis. The finding of pancreatic NF-
B
activation in several experimental models of pancreatitis indicates
that NF-
B activation may be an important common mechanism in the
development of acute pancreatitis.
We observed a strong AP-1 response already at 1 h after
taurocholate infusion. However, in contrast to NF-B, AP-1 binding activity in the pancreas did not further increase at 6 h after taurocholate infusion. Different homo- and heterodimeric AP-1 complexes
can differently regulate transcription of target genes (50). Our supershift assay demonstrated the presence of
c-Fos, Jun-B, and Fra-2 in the AP-1 complexes activated in response to taurocholate infusion. AP-1 activation in the pancreas has not been
described previously. So far, two studies reported pancreatic induction
of the protoncogenes c-fos and c-jun during
cerulein-induced pancreatitis in mice (13) and rats
(10). The role of the different AP-1 subunits we found
activated in taurocholate pancreatitis in the expression of genes
mediating the inflammatory response remains to be determined.
We measured the pancreatic mRNA expression of proinflammatory cytokines
(IL-6 and TNF-), chemokines (KC and MCP-1), and iNOS in saline- and
taurocholate-infused rats. Promoters of all of these genes contain
binding sites for NF-
B and AP-1 (4, 33, 34, 40). Our
results show that taurocholate pancreatitis is associated with an early
upregulation in the head of the pancreas of all the mediators studied
except TNF-
. Because TNF-
induction has been reported for other
models of pancreatitis (9, 13, 16, 38), this finding may
be dependent on the specific characteristics of taurocholate-induced
pancreatitis. It also may be that TNF-
activation in this model is
delayed beyond the 6-h observation period. Of particular interest is
the strong upregulation we observed for the chemokine KC, a potent
neutrophil chemoattractant (30).
In contrast to other experimental models of acute pancreatitis, in
which the pancreas is entirely affected, the intraductal infusion of
taurocholate renders a regional affectation mostly limited to the
pancreatic head. Such localized damage is an inherent characteristic of
this model. This and previous studies (25, 31, 45, 46)
show more pronounced morphological injury, microcirculatory changes,
trypsin activation, and neutrophil infiltration in the pancreatic head
than in the tail. In this study, we found a localized activation of
NF-B and expression of inflammatory molecules in the pancreatic
head. The correlation between regional activation of NF-
B (and the
cytokines regulated by NF-
B) and the parameters that reflect
pancreatic damage adds new evidence to the key role of this
transcription factor in tissue injury. In contrast to NF-
B, there
was not much difference in AP-1 activation between the pancreatic head
and tail. Also, the increase in AP-1-binding activity was similar in
saline- and taurocholate-infused rats. This suggests that, although
AP-1 may be necessary for the full activation of specific inflammatory
molecules, it is not sufficient by itself to do so.
To establish whether the changes observed in taurocholate-induced
pancreatitis were dependent on NF-B activity, we used the NF-
B
inhibitor NAC. This antioxidant is well known to inhibit NF-
B
activation in vivo (15, 17, 51). In agreement with what we
previously reported for cerulein pancreatitis (17), NAC
markedly inhibited NF-
B activation in the 6 h of taurocholate pancreatitis, with significant amelioration of the local inflammatory response (neutrophil infiltration and expression of inflammatory molecules). This finding supports the idea that NF-
B activation is a
key event in triggering the local cascade of inflammatory mediators
associated with acute pancreatitis. NAC did not affect serum amylase
and lipase levels at 6 h after taurocholate infusion. This finding
may be explained by the multifactorial nature of pancreatic injury
associated with the taurocholate model, one of the factors being
"mechanical" injury. Once the integrity of the pancreatic tissue is
disrupted by the direct action of the taurocholic bile salt, pancreatic
enzymes may reach the circulation independently of any mechanism
regulating the inflammatory process. The absence of such mechanical
factors in cerulein pancreatitis may account for the observation of a
greater effect of NAC on serum amylase and lipase in the cerulein model
(17).
It is of note that the beneficial effects of NF-B and cytokine
inhibition may also be pronounced at later stages of
taurocholate-induced pancreatitis. Inhibition of NF-
B with another
antioxidant, pyrrolidinedithiocarbamate, has been reported to improve
the survival of rats with taurocholate pancreatitis
(42). Our study focused on the early events in this model.
Finally, our data provide characterization of some molecular events in
the pancreatic response to saline intraductal infusion. Saline infusion
into the pancreatic duct is not a harmless procedure (2, 31,
45). The hydrostatic pressure increase is one of several factors
that can trigger the inflammatory reaction in the pancreas
(32). In this regard, intraductal saline infusion may be
extrapolated to the endoscopic retrograde cholangiopancreatography used
in humans that is associated with a risk of pancreatitis (18,
29). In the present study, although saline infusion did not
cause the necrohemorrhagic changes as observed after taurocholate infusion, it did induce significant hyperamylasemia and hyperlipasemia, inflammatory cell infiltration, and trypsin activation in the head of
the pancreas. Furthermore, we observed saline-induced activation of
NF-B and some cytokines/chemokines (e.g., IL-6, KC, and MCP-1) in
response to saline infusion. A particularly strong activation was
observed for AP-1, which was comparable to that induced by
taurocholate. These results indicate that an increase in
intrapancreatic hydrostatic pressure in itself can trigger some
pathways initiating an inflammatory response.
In summary, our study demonstrated that taurocholate infusion results
in activation of the transcription factors NF-B and AP-1 and the
expression of proinflammatory cytokines/chemokines and iNOS in the
pancreas. NF-
B (but not AP-1) activation and upregulation of
proinflammatory molecules were more pronounced in the head than in the
tail segment of the pancreas. The localized responses correlated with
the severity of the lesion, including morphological changes, neutrophil
infiltration, and pancreatic trypsin activation. Inhibition of NF-
B
activation resulted in amelioration of the local inflammatory response.
Intraductal saline infusion also caused morphological and molecular
changes that reflect pancreatic injury, although much milder compared
with taurocholate infusion. The results indicate that activation of transcription factors and inflammatory molecule expression is a common
mechanism in the development of acute pancreatitis in different models
of pancreatitis.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by Grant FIS 98/5072 from the Ministry of Health, Spain (to E. Vaquero), the Department of Veterans Affairs, The Research Center for Alcoholic Liver and Pancreatic Diseases (NIAAA Grant P-50-AA-11999), and by the Andy Barnes Family Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: E. Vaquero, VA Greater Los Angeles Healthcare System, West Los Angeles, Bldg. 258, Rm. 331, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: evaquero{at}ucla.edu).
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 29 June 2000; accepted in final form 24 January 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adler, G,
Hupp T,
and
Kern HF.
Course and spontaneous regression of acute pancreatitis in the rat.
Virchows Arch Pathol Anat
382:
31-47,
1979[ISI][Medline].
2.
Aho, HJ,
Koskensalo SM,
and
Nevalainen TJ.
Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis.
Scand J Gastroenterol
15:
411-416,
1980[ISI][Medline].
3.
Baeuerle, PA,
and
Baichwal VR.
NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules.
Adv Immunol
65:
111-137,
1997[ISI][Medline].
4.
Ben-Baruch, A,
Michiel DF,
and
Oppenheim JJ.
Signals and receptors involved in recruitment of inflammatory cells.
J Biol Chem
270:
11703-11706,
1995
5.
Berney, T,
Gasche Y,
Robert J,
Jenny A,
Mensi N,
Grau G,
Vermeulen B,
and
Morel P.
Serum profiles of interleukin-6, interleukin-8, and interleukin-10 in patients with severe and mild acute pancreatitis.
Pancreas
18:
371-377,
1999[ISI][Medline].
6.
Bhatia, M,
Brady M,
Shokuhi S,
Christmas S,
Neoptolemos JP,
and
Slavin J.
Inflammatory mediators in acute pancreatitis.
J Pathol
190:
117-125,
2000[ISI][Medline].
7.
Denham, W,
Denham D,
Yang J,
Carter G,
MacKay S,
Moldawer LL,
Carey LC,
and
Norman J.
Transient human gene therapy: a novel cytokine regulatory strategy for experimental pancreatitis.
Ann Surg
227:
812-820,
1998[ISI][Medline].
8.
Denham, W,
Yang J,
Fink GW,
Denham D,
Carter G,
Ward K,
and
Norman J.
Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis.
Gastroenterology
113:
1741-1746,
1997[ISI][Medline].
9.
Dunn, JA,
Li C,
Ha T,
Kao RL,
and
Browder W.
Therapeutic modification of nuclear factor kappa B binding activity and tumor necrosis factor-alpha gene expression during acute biliary pancreatitis.
Am J Surg
63:
1036-1044,
1997.
10.
Ferrara, C,
Gress TM,
Mueller-Pillasch F,
Lutz MP,
Weidenbach H,
Poletti A,
Lerch MM,
Del Favero G,
and
Adler G.
Expression of the protooncogene jun is induced in the rat pancreas by cerulein infusion.
Pancreas
15:
160-167,
1997[ISI][Medline].
11.
Fink, GW,
and
Norman JG.
Specific changes in the pancreatic expression of the interleukin 1 family of genes during experimental acute pancreatitis.
Cytokine
9:
1023-1027,
1997[ISI][Medline].
12.
Frossard, JL,
Saluja AK,
Bhagat L,
Lee HS,
Bhatia M,
Hofbauer B,
and
Steer ML.
The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury.
Gastroenterology
116:
694-701,
1999[ISI][Medline].
13.
Fu, K,
Sarras MPJ,
De Lisle RC,
and
Andrews GK.
Expression of oxidative stress-responsive genes and cytokine genes during caerulein-induced acute pancreatitis.
Am J Physiol Gastrointest Liver Physiol
273:
G696-G705,
1997
14.
Ghosh, S,
May MJ,
and
Kopp EB.
NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu Rev Immunol
16:
225-260,
1998[ISI][Medline].
15.
Gius, D,
Botero A,
Shah S,
and
Curry HA.
Intracellular oxidation/reduction status in the regulation of transcription factors NF-kappaB and AP-1.
Toxicol Lett
106:
93-106,
1999[ISI][Medline].
16.
Gukovskaya, AS,
Gukovsky I,
Zaninovic V,
Song M,
Sandoval D,
Gukovsky AS,
and
Pandol SJ.
Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-. Role in regulating cell death and pancreatitis.
J Clin Invest
100:
1853-1862,
1997
17.
Gukovsky, I,
Gukovskaya AS,
Blinman TA,
Zaninovic V,
and
Pandol SJ.
Early NF- B activation is associated with hormone-induced pancreatitis.
Am J Physiol Gastrointest Liver Physiol
275:
G1402-G1414,
1998
18.
Halme, L,
Doepel M,
von Numers H,
Edgren J,
and
Ahonen J.
Complications of diagnostic and therapeutic ERCP.
Ann Chir Gynaecol
88:
127-131,
1999[ISI][Medline].
19.
Han, B,
and
Logsdon CD.
Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappaB activation.
Am J Physiol Cell Physiol
277:
C74-C82,
1999
20.
Heath, DI,
Cruickshank A,
Gudgeon M,
Jehanli A,
Shenkin A,
and
Imrie CW.
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].
21.
Hughes, CB,
Grewal HP,
Gaber LW,
Kotb M,
El-din AB,
Mann L,
and
Gaber AO.
Anti-TNF alpha therapy improves survival and ameliorates the patholphysiologic sequelae in acute pancreatitis in the rat.
Am J Surg
171:
274-280,
1996[ISI][Medline].
22.
Karin, M,
Liu Z,
and
Zandi E.
AP-1 function and regulation.
Curr Opin Cell Biol
9:
240-246,
1997[ISI][Medline].
23.
Kawabata, S,
Miura T,
Morita T,
Kato H,
Fujikawa K,
Iwanaga S,
Takada T,
Kimura T,
and
Sakakibara S.
Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin.
Eur J Biochem
172:
17-25,
1988[Abstract].
24.
Kingsnorth, A.
Role of cytokines and their inhibitors in acute pancreatitis.
Gut
40:
1-4,
1997
25.
Kusterer, K,
Enghofer M,
Zendler S,
Blochle C,
and
Usadel KH.
Microcirculatory changes in sodium taurocholate-induced pancreatitis in rats.
Am J Physiol Gastrointest Liver Physiol
260:
G346-G351,
1991
26.
Lampel, M,
and
Kern HF.
Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue.
Virchows Arch Pathol Anat
373:
97-117,
1977[ISI][Medline].
27.
Lankisch, PG,
and
Ihse I.
Bile-induced acute experimental pancreatitis.
Scand J Gastroenterol
22:
257-260,
1987[ISI][Medline].
28.
Leser, HG,
Gross V,
Scheibenbogen C,
Heinisch A,
Salm R,
Lausen M,
Ruckauer K,
Andreesen R,
Farthmann EH,
and
Scholmerich J.
Elevation of serum interleukin-6 concentration precedes acute-phase response and reflects severity in acute pancreatitis.
Gastroenterology
101:
782-785,
1991[ISI][Medline].
29.
Loperfido, S,
Angelini G,
Chilovi F,
Costan F,
De Berardinis F,
Bernardin M,
Ederle A,
Fina P,
and
Fratton A.
Major early complications from diagnostic and therapeutic ERCP: a prospective multicenter study.
Gastrointest Endosc
48:
1-10,
1998[ISI][Medline].
30.
Luster, AD.
Chemokines-chemotactic cytokines that mediate inflammation.
N Engl J Med
338:
436-445,
1998
31.
Luthen, R,
Grendell JH,
Niederau C,
and
Haussinger D.
Trypsinogen activation and glutathione content are linked to pancreatic injury in models of biliary acute pancreatitis.
Int J Pancreatol
24:
193-202,
1998[ISI][Medline].
32.
Luthen, R,
Niederau C,
Niederau M,
Ferrell LD,
and
Grendell JH.
Influence of ductal pressure and infusates on activity and subcellular distribution of lysosomal enzymes in the rat pancreas.
Gastroenterology
109:
573-581,
1995[ISI][Medline].
33.
Marks-Konczalik, J,
Chu SC,
and
Moss J.
Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites.
J Biol Chem
273:
22201-22208,
1998
34.
Martin, T,
Cardarelli PM,
Parry GC,
Felts KA,
and
Cobb RR.
Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.
Eur J Immunol
27:
1091-1097,
1997[ISI][Medline].
35.
Molero, X,
Guarner F,
Salas A,
Mourelle M,
Puig V,
and
Malagelada JR.
Nitric oxide modulates pancreatic basal secretion and response to cerulein in the rat: effects in acute pancreatitis.
Gastroenterology
108:
1855-1862,
1995[ISI][Medline].
36.
Norman, J.
The role of cytokines in the pathogenesis of acute pancreatitis.
Am J Surg
175:
76-83,
1998[ISI][Medline].
37.
Norman, J,
Franz MG,
Messina J,
Riker A,
Fabri PJ,
Rosemurgy AS,
and
Gower WRJ
Interleukin-1 receptor antagonist decreases severity of experimental acute pancreatitis.
Surgery
117:
648-655,
1995[ISI][Medline].
38.
Norman, JG,
Fink GW,
and
Franz MG.
Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression.
Arch Surg
130:
966-970,
1995[Abstract].
39.
Pandol, SJ,
Periskic S,
Gukovsky I,
Zaninovic V,
Jung Y,
Zong Y,
Solomon TE,
Gukovskaya AS,
and
Tsukamoto H.
Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide.
Gastroenterology
117:
706-716,
1999[ISI][Medline].
40.
Roebuck, KA,
Carpenter LR,
Lakshminarayanan V,
Page SM,
Moy JN,
and
Thomas LL.
Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-kappaB.
J Leukoc Biol
65:
291-298,
1999[Abstract].
41.
Sandoval, D,
Gukovskaya AS,
Reavey P,
Gukovsky S,
Sisk A,
Braquet P,
Pandol SJ,
and
Poucell-Hatton S.
The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis.
Gastroenterology
111:
1081-1091,
1996[ISI][Medline].
42.
Satoh, A,
Shimosegawa T,
Fujita M,
Kimura K,
Masamune A,
Koizumi M,
and
Toyota T.
Inhibition of nuclear factor-kappaB activation improves the survival of rats with taurocholate pancreatitis.
Gut
44:
253-258,
1999
43.
Schmid, RM,
and
Adler G.
Cytokines in acute pancreatitis-new pathophysiological concepts evolve.
Eur J Gastroenterol Hepatol
11:
125-127,
1999[ISI][Medline].
44.
Steinle, AU,
Weidenbach H,
Wagner M,
Adler G,
and
Schmid RM.
NF-B/Rel activation in cerulein pancreatitis.
Gastroenterology
116:
420-430,
1999[ISI][Medline].
45.
Telek, G,
Scoazec JY,
Chariot J,
Ducroc R,
Feldmann G,
and
Roz C.
Cerium-based histochemical demonstration of oxidative stress in taurocholate-induced acute pancreatitis in rats. A confocal laser scanning microscopic study.
J Histochem Cytochem
47:
1201-1212,
1999
46.
Werner, J,
Dragotakes SC,
Fernandez-del Castillo C,
Rivera J,
Ou J,
Rattner DW,
Fischman AJ,
and
Warshaw AL.
Technetium-99m-labeled white blood cells: a new method to define the local and systemic role of leukocytes in acute experimental pancreatitis.
Ann Surg
227:
86-94,
1998[ISI][Medline].
47.
Werner, J,
Rivera J,
Fernandez-del Castillo C,
Lewandrowski KB,
Adrie C,
Rattner DW,
and
Warshaw AL.
Differing roles of nitric oxide in the pathogenesis of acute edematous versus necrotizing pancreatitis.
Surgery
12:
23-30,
1997.
48.
Werner, J,
Z'graggen K,
Fernandez-del Castillo C,
Lewandrowski KB,
Compton CC,
and
Warshaw AL.
Specific therapy for local and systemic complications of acute pancreatitis with monoclonal antibodies against ICAM-1.
Ann Surg
229:
834-840,
1999[ISI][Medline].
49.
Whitmarsh, AJ,
and
Davis RJ.
Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways.
Mol Med
74:
589-607,
1996.
50.
Wisdom, R.
AP-1: one switch for many signals.
Exp Cell Res
253:
180-185,
1999[ISI][Medline].
51.
Wulczyn, FG,
Krappmann D,
and
Scheidereit C.
The NF-B/Rel and I-
B gene families: mediators of immune response and inflammation.
J Mol Med
74:
749-769,
1996[ISI][Medline].
52.
Zaninovic, V,
Gukovskaya AS,
Gukovsky I,
Mouria M,
and
Pandol SJ.
Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells.
Am J Physiol Gastrointest Liver Physiol
279:
G666-G676,
2000