Division of Gastroenterology, Geneva University Hospital, 1211 Geneva 14, Switzerland
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ABSTRACT |
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Although the pancreatic heat shock
response has already been reported to confer protective effects during
experimental pancreatitis, the mechanism of action remains unknown. We
investigated the effects of hyperthermia in cerulein-induced
pancreatitis. Heat shock protein 70 (HSP70) expression in rats was
induced by a 20-min period of water immersion (42°C). The severity of
pancreatitis as well as the pancreatic expression of cytokines,
nuclear factor-B (NF-
B), and inhibitory factor
B-
(I
B-
) were evaluated in the presence and absence of hyperthermia.
We found that hyperthermia resulted in time-dependent expression of
HSP70 within the pancreas associated with a reduction in the severity
of acute pancreatitis. Tumor necrosis factor-
and intercellular
adhesion molecule-1 expression was significantly reduced in the
presence of hyperthermia. Moreover, NF-
B activity was delayed in the
presence of hyperthermia whereas I
B-
was stabilized in the
cytoplasm. These results suggest that hyperthermia decreases the
severity of cerulein-induced pancreatitis by decreasing cytokine
expression in the pancreas through the modulation of NF-
B activity.
cytokines; heat shock proteins; intercellular adhesion molecule-1
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INTRODUCTION |
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EXPOSURE OF PROKARIOTIC and eukaryotic cells to hyperthermia induces a rapid and constant modification of cell metabolism called the heat shock response (17, 18). This response is mainly the consequence of the modified expression of stress proteins that were first described by Ritossa (22) 39 years ago. Raising the temperature of Drosophila above its physiological norm decreased the expression of several genes that were active before heat exposure and increased the expression of other genes encoding for a group of proteins referred as the heat shock proteins (HSPs). The main role of HSPs is of thermotolerance; a brief sublethal heat exposure of isolated cells induces quick expression of HSPs, conferring total protection against a subsequent but lethal exposure to hyperthermia. Additionally, HSPs also protect cells from other sources of stress such as endotoxins (30) and oxygen radicals (1, 30). Recently, we (9) also showed that HSPs prolong the in vivo survival time of rats suffering from anoxia.
Among the HSPs, HSP70 protects against experimental pancreatitis. The
protective role of hyperthermia during experimental pancreatitis has
already been investigated (29). Wagner et al. (29) demonstrated that hyperthermia partially protected
acute pancreatitis by reducing acinar cell necrosis. Other studies
(11, 12) showed that HSP70 expression after hyperthermia
decreased the severity of cerulein-induced acute pancreatitis by
lowering intrapancreatic trypsin activity. However, the protective
mechanism of HSP70 is not completely understood. For example, whether
hyperthermia decreases intercellular adhesion molecule-1 (ICAM-1) and
tumor necrosis factor- (TNF-
) expression in pancreas after
cerulein is unknown.
Among the mechanisms involved in the protection conferred by
hyperthermia, a modification of nuclear factor-B (NF-
B) activity might be important. Indeed, the transcription NF-
B is a pleiotropic regulator of many genes involved in inflammatory responses. NF-
B is
rapidly activated after the induction of acute pancreatitis by cerulein
(14-16). NF-
B activates genes encoding for
proinflammatory cytokines (13, 16), such as TNF-
and
interleukin-6 (IL-6), which in turn aggravate the severity of
pancreatitis (19). Additionally, Rossi et al.
(23) demonstrated that HSPs inhibit NF-
B activation through activation of the heat shock transcription factor.
The aim of our study was to better define the cytoprotective action of
hyperthermia in a well-defined model of cerulein-induced acute
pancreatitis. This experimental model induces edematous pancreatitis,
the severity of which is correlated with pancreatic TNF-
concentrations (19), endothelial ICAM-1 expression
(10), and neutrophil infiltration (2, 10,
25). Because previous studies (27) showed that
hyperthermia can inhibit the activation of NF-
B and NF-
B is known
as an activator of genes encoding for proinflammatory cytokines such as
IL-6 and TNF-
, which can in turn upregulate the expression of
adhesion molecules (27), we hypothesized that hyperthermia
might abolish NF-
B nuclear migration and decrease TNF-
and ICAM-1
expression with a concomitant reduction of pancreatic injury.
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METHODS |
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Animals. All experiments were performed according to protocols approved by institutional animal care and the veterinary office. Male Wistar rats (75-125 g), obtained from BRL, were bred and housed in standard cages in a thermostated (23 ± 2°C) environment with 12:12-h light-dark cycles. The rats ate standard laboratory chow, drank water ad libitum, and were then randomly assigned to control or treated groups.
Drugs. Cerulein, the decapeptide analog of the pancreatic secretagogue, was purchased from Research Plus (Bayonne, NJ). Superfrost Plus slides were provided by Fisher Scientific (Pittsburgh, PA). All other chemicals and reagents were purchased from Sigma Chemical (Basel, Switzerland).
Induction of pancreatitis.
Rats were intraperitoneally injected twice at a 1-h interval with a
supramaximally stimulating dose (10 µg/kg) of cerulein to elicit
acute pancreatitis (see Fig. 1). Control
rats received similar injections of saline solution. The animals were
killed 2 or 4 h after the last cerulein or saline injection with
pentobarbital sodium injection (50 mg/kg ip).
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Induction of hyperthermia. To produce hyperthermia rats were immersed in hot water (42 ± 2°C), and rectal temperature was monitored. Whole body temperature slowly increased over 25 min to reach 42°C and remained at this level for 20 min. To determine the time course expression of hyperthermia-induced HSP70 expression within the pancreas, rats were killed every 4 h over 24 h.
Blood and tissue preparation.
Blood and pancreatic tissue were processed as described previously
(10). Briefly, at the time of death blood was withdrawn from the heart and centrifuged, and the serum was kept at 80°C until assayed. Three pieces of pancreas were collected for histological studies, myeloperoxidase (MPO) quantitation (which reflects the amount
of neutrophils sequestered within the pancreas), and NF-
B gel shift
assay. For HSP70 and ICAM-1 quantification, pancreas samples were
homogenized in 1 ml of phosphate buffer (20 mM, pH 7.4).
Quantification of pancreatic injury.
Serum amylase activity was measured as described by Pierre et al.
(21) using 4,6-ethylidene
(G1)-p-nitrophenyl
(G1)-D-maltoheptoside (Sigma Chemical, St.
Louis, MO) as the substrate. The extent of pancreatic edema was
quantitated by measuring tissue water content; pancreatic tissue was
weighed before and after desiccation at 95°C for 24 h. The
difference between the wet and dry tissue weights was calculated and
expressed as a percentage of the tissue wet weight.
SDS-PAGE and Western blot transfer for HSP70 protein, ICAM-1, and
IB proteins.
To detect HSP70 protein, Western blot was performed according to the
method of Laemmli (16a) using minigels (Bio-Rad,
Zurich, Switzerland). Proteins (10 µg) were loaded in each
lane. After gel electrophoresis, proteins were transferred to
nitrocellulose membrane according to Towbin et al. (28).
The membrane was then incubated for 2 h with primary antibody
(mouse monoclonal anti-HSP70, Sigma Chemical). After rinsing in
PBS-Tween, the membrane was incubated with secondary antibody
(anti-mouse IgG, Bio-Rad) for 1 h at room temperature. The same
procedure was used to detect ICAM-1 expression (30 µg of protein were
loaded in each lane) within the pancreas using mouse anti-ICAM-1
monoclonal antibody (mouse CD54, R&D Systems). For I
B measurement,
cellular protein extracts were diluted in SDS-PAGE loading buffer and
then resolved by conventional electrophoresis. The blot was incubated
with a primary antibody against I
B
for 2 h (Santa Cruz
Biotechnology), and after washing the blot was incubated with a
secondary antibody (goat anti-mouse IgG, Bio-Rad).
Nuclear protein extracts.
To allow NF-B measurement, nuclear protein extracts were prepared
essentially as described by Dignam et al. (7). A 150- to
200-mg pancreatic tissue sample was lysed on ice in a hypotonic buffer
by 20 or 25 strokes in a glass Dounce homogenizer. The hypotonic buffer
was supplemented with phenylmethylsulfonyl fluoride (PMSF, 1 mM),
dithiothreitol (DTT, 1 mM), and protease inhibitors, including
pepstatin (5 µg/ml), leupeptin (5 µg/ml), and aprotinin (5 µg/ml). The homogenate was left on ice for 15-20 min, and
Nonidet P-40 was added to a final concentration of 0.3-0.4%
(vol/vol). 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 I
B, and the nuclear pellet was resuspended in the
high-salt buffer containing 20 mM HEPES (pH 7.6), 25% (vol/vol)
glycerol, 0.42 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM
glycerophosphate, 10 mM Na2MgSO4, 1 mM DTT, and
1 mM PMSF. After being rotated at 4°C overnight, the nuclear
membranes were pelleted by microcentrifugation for 10 min, and the
clear supernatant (nuclear extract) was aliquoted and stored at
80°C. The protein concentration in the nuclear extract was
determined by the Bio-Rad protein assay.
Electrophoretic mobility shift assay.
To measure NF-B binding activity, aliquots of nuclear extracts with
equal amount of protein (10 µg) were mixed 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 of poly(dI/dC). After aliquots were equilibrated
on ice for 5 min, binding reactions were started by the addition of
20-60,000 counts/min 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'-GCAGAGGGGACTTTCCGAGA containing a
B binding motif was annealed to the complementary oligonucleotide with a 5'-G overhang and end-labeled using Klenow DNA
polymerase I. Samples were electrophoresed at room temperature in
0.5 × TBE buffer (1 × TBE = 89 mM Tris base, 89 mM
boric acid, and 2 mM EDTA) on nondenaturing 4.5% polyacrylamide gel at
200 V. Gels were dried and directly exposed at
80°C to Kodak RX
film with intensifying screens. The intensity of the bands on the gel was quantified using the image analysis system from Zeiss (Zurich, Switzerland)
TNF- assay.
TNF-
was quantitated using a commercially available ELISA kit
(Endogen, Woburn, MA). Pancreas tissue cytokine levels were measured by
homogenizing a sample of freshly obtained tissue in 2 ml of phosphate
buffer (20 mM, pH 7.4), subjecting it to centrifugation (14,000 g for 5 min at 4°C), and quantitating TNF-
in the
resulting supernatant. Results were expressed as picograms per
microgram of DNA in the sample.
Morphology. Sections of pancreatic tissues were rapidly removed at the time of death, fixed, embedded in paraffin, and sectioned (5 µm). After staining with hematoxylin and eosin, the sections were examined by an experienced morphologist who was not aware of the sample identity. The extent of acinar cell necrosis was quantitated by computer-assisted morphometry as previously described (10) and expressed as a percentage of total acinar tissue.
Analysis of data. The results are expressed as means ± SE of values obtained from at least three different determinations. The significance of changes was evaluated using Student's t-test when data included two groups or ANOVA when three or more groups were compared.
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RESULTS |
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Hyperthermia induced intrapancreatic HSP70 protein expression.
Whole body hyperthermia resulted in time-dependent expression of HSP70
within the pancreas with a peak occurring 12 h after the heating
procedure (Fig. 2). For the following
experiments, all animals were studied at the peak of HSP70 expression
within the pancreas (12 h after hyperthermia) because the
cytoprotective effects of hyperthermia were maximal at that time.
Twelve hours after whole body hyperthermia, rats were killed and in
vitro amylase secretion from acini was studied as a function of
cerulein concentration. The pattern of secretion was identical to the
pattern observed in control animals, indicating that the receptor
remained functionally intact after hyperthermia (data not shown).
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Effects of hyperthermia on severity of cerulein-induced acute
pancreatitis.
The severity of the cerulein-induced acute pancreatitis was assessed by
serum amylase concentration, edema within the gland, the amount of
neutrophils sequestered within the gland as reflected by MPO activity,
and acinar cell necrosis. Supramaximal stimulation of rats with
cerulein increased concentrations of serum amylase, edema within the
gland, and acinar cell necrosis 3 and 5 h after the start of
cerulein injection (Fig. 3). Cerulein
injections, in the absence of hyperthermia, concomitantly increased
intrapancreatic MPO activity. In contrast, when rats were exposed to
hyperthermia 12 h before cerulein injections, the severity of
acute pancreatitis decreased. Hyperthermia by itself had no effect on
MPO activity in control rats.
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Intrapancreatic TNF- concentrations in cerulein-treated rats in
the presence or absence of hyperthermia.
Injection of cerulein increased pancreatic TNF-
concentrations over
time (Fig. 4). Pancreatic TNF-
concentrations also increased in rats previously exposed to
hyperthermia before cerulein injections. However, the peak of TNF-
was higher in the absence of hyperthermia (3.56 ± 0.3 pg/µg DNA
at 3 h; 6.44 ± 0.48 pg/µg DNA at 5 h) than in the
presence of hyperthermia (1.42 ± 0.2 pg/µg DNA at 3 h; 2.56 ± 0.26 pg/µg DNA at 5 h) (P < 0.01).
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Intrapancreatic ICAM-1 expression in cerulein-treated rats in
presence or absence of hyperthermia.
Cerulein injections induced a small but significant rise of ICAM-1
expression in the absence of hyperthermia (Fig.
5) 3 h after the start of cerulein
injection and a more significant rise after 5 h. The ICAM-1
expression was mainly located on the endothelium surface (data not
shown). When rats had been previously exposed to hyperthermia,
pancreatic ICAM-1 content was lower. The optical density of the ICAM-1
band was 2.91 times higher in cerulein-treated animals at 3 h and
3.24 times higher at 5 h compared with pancreas isolated from
animals injected with saline. ICAM-1 expression within the pancreas of
cerulein-injected animals previously exposed to hyperthermia was
slightly increased (optical density 1.15 times higher than in animals
injected with saline at 3 h and 1.77 times higher at 5 h),
but the increase was significantly lower than the increase observed in
pancreas isolated from animals injecting with cerulein in the absence
of hyperthermia (P < 0.02).
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NF-B binding activity in normal and cerulein-treated rats in
presence or absence of hyperthermia.
In normal animals, there was no detection of NF-
B activity and
hyperthermia alone did not induce the activation of NF-
B (Fig.
6). Cerulein injections increased NF-
B
binding activity over time in the absence of hyperthermia. NF-
B
binding activity rapidly increased 30 min after cerulein injections and
peaked at 60 min. Cerulein injection in rats previously exposed to
hyperthermia completely abolished the increase of NF-
B binding
activity within the first 45 min. However, the activity appeared at 45 min and reached a maximum by 90 min. Thus after cerulein treatment,
NF-
B activity was significantly modified by hyperthermia.
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DISCUSSION |
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Our study clearly shows that hyperthermia is associated with a
time-dependent increase of pancreatic HSP70 expression, which peaked
12 h after heat stress. Concomitantly, hyperthermia protects against cerulein-induced acute pancreatitis as evidenced by decreased serum amylase concentration, pancreatic edema, and acinar cell necrosis
extent. Moreover, TNF- and ICAM-1 expression, two parameters correlated with the severity of cerulein-induced pancreatitis (10, 20), were significantly reduced in the presence of
hyperthermia. Finally, the nuclear migration of NF-
B was delayed by
hyperthermia whereas the NF-
B-I
B-
complex was stabilized in
the cytoplasm. These results suggest that hyperthermia decreases the
severity of cerulein-induced pancreatitis by decreasing cytokine
expression in the pancreas through the modulation of NF-
B activity.
Because in this model we clearly demonstrated that the CCK-receptor
binding characteristics after hyperthermia are unaltered by in vitro
amylase secretion study (12), the beneficial effects due
to hyperthermia are not related to a decreased binding of cerulein to
the receptor. In our study, TNF- was upregulated in the pancreas
during cerulein hyperstimulation as previously shown by Gukovskaya et
al. (14). TNF-
levels rise in a time-dependent manner
in animals injected with cerulein, whereas these levels were
significantly lower when rats were previously exposed to hyperthermia.
The deleterious role of TNF-
in inducing organ injury has already
been investigated. TNF-
might promote organ injury by upregulating
ICAM-1 (19), an important adhesion molecule involved in
the adhesion of circulating activated inflammatory cells to the
microvascular endothelial surfaces and in the leukocyte sequestration
within areas of pancreatic injury and inflammation (10).
In our study, ICAM-1 expression was markedly elevated during
cerulein-induced pancreatitis, as was MPO content, which reflects the
sequestration of inflammatory cells. We also found that both
ICAM-1 expression and MPO content increases were significantly attenuated in the presence of hyperthermia. Thus rats exposed to
hyperthermia are partially protected from cerulein-induced pancreatitis, because both TNF-
and ICAM-1 expression were blunted with a concomitant decrease of neutrophil recruitment into the pancreas, which plays an important role in the development of injury
(2, 10, 25).
As shown by Gukovsky et al. (15), NF-B was rapidly
activated by cerulein in the rat pancreas. The activity peaked by 45 min after the start of cerulein injections (15).
Interestingly, the pattern of NF-
B activity was different in
pancreatic tissue expressing a high amount of HSP70 protein. Indeed, in
the presence of hyperthermia, NF-
B activity was first blunted within
the first 45 min while its activity appeared only by 60 min. In the
absence of hyperthermia, NF-
B activity peaked at 60 min in the
nucleus while the I
B
signal disappeared in the cytoplasm.
However, in the presence of hyperthermia, NF-
B nuclear translocation
was delayed and was probably associated with stabilization of I
B
in the cytoplasm. Thus the delayed NF-
B activity was likely to
decrease cytokine expression in the pancreas, which in turn reduced the
severity of acute pancreatitis.
Indeed, HSPs might directly interfere with NF-B, which is a key
regulator of inducting cytokines such as TNF-
, IL-1, and IL-6
(13). Heat exposure is also known to inhibit
cytokine-mediated inducible nitric oxide synthase gene expression
through the activation of the same transcriptional regulator NF-
B
(26). Several studies found (23, 24) that the
heat shock response inhibits cytokine-mediated NF-
B nuclear
translocation in human isolated cells. The heat shock response also
inhibits cytokine-mediated I
B degradation by a chaperone
cytoprotective mechanism in the rat liver (4, 5). Lastly,
Curry et al. (6) demonstrated that HSPs transiently inhibited radiation-induced NF-
B DNA binding activity by preventing I
B kinase activation. Thus the increased expression of HSP in our
model might be responsible for the delayed NF-
B activity observed in
the presence of hyperthermia.
Our data suggest that the heat shock response protects against
cerulein-induced pancreatitis by interfering with transcriptional mechanisms involving, at least in this study, the NF-B-I
B
pathway. However, it is possible that HSPs may act through other
transcription factors to influence the severity of pancreatitis.
Indeed, HSPs were shown (3) to modulate the cellular
activator protein-1, a transcription factor involved in multiple
aspects of cell regulation (8). The molecular protective
mechanism of hyperthermia is not fully understood; further study is
needed to comprehend it.
In conclusion, our study shows that hyperthermia decreases cytokine
expression in the pancreas with a concomitant reduction of the severity
of acute pancreatitis. Because HSP70 is highly expressed within the
pancreas 12 h after hyperthermia and HSP protein has been shown to
decrease NF-B activity, the delayed activity of NF-
B observed in
our study is likely to stem from HSPs. However, the mechanisms involved
between NF-
B and HSP70 and between NF-
B and cytokine expression
need further study. Our study suggests that the heat shock pathway is
able to simultaneously switch on cytoprotective genes and downregulate
genes encoding for proinflammatory cytokines such as TNF-
.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Swiss National Science Foundation (32.63618.00).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Frossard, Division of Gastroenterology, Geneva Univ. Hospital, 1211 Geneva 14, Switzerland (E-mail: jean-louis.frossard{at}hcuge.ch).
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 6 July 2000; accepted in final form 5 January 2001.
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