Effect of hyperthermia on NF-kappaB binding activity in cerulein-induced acute pancreatitis

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Article

Reference

Effect of hyperthermia on NF-kappaB binding activity in cerulein-induced acute pancreatitis

FROSSARD, Jean-Louis, PASTOR, Catherine, HADENGUE, Antoine

Abstract

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 degrees C). The severity of pancreatitis as well as the pancreatic expression of cytokines, nuclear factor-kappaB (NF-kappaB), and inhibitory factor kappaB-alpha (IkappaB-alpha) 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-alpha and intercellular adhesion molecule-1 expression was significantly reduced in the presence of hyperthermia. Moreover, NF-kappaB activity was delayed in the presence of hyperthermia whereas IkappaB-alpha was stabilized in the cytoplasm. These results suggest that hyperthermia decreases the severity of cerulein-induced pancreatitis by decreasing cytokine [...]

FROSSARD, Jean-Louis, PASTOR, Catherine, HADENGUE, Antoine. Effect of hyperthermia on NF-kappaB binding activity in cerulein-induced acute pancreatitis. American Journal of

Physiology. Gastrointestinal and Liver Physiology , 2001, vol. 280, no. 6, p. G1157-62

PMID : 11352808

Available at:

http://archive-ouverte.unige.ch/unige:73842

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Effect of hyperthermia on NF- ␬ B binding activity in cerulein-induced acute pancreatitis

JEAN LOUIS FROSSARD, CATHERINE M. PASTOR, AND ANTOINE HADENGUE Division of Gastroenterology, Geneva University Hospital, 1211 Geneva 14, Switzerland

Received 6 July 2000; accepted in final form 5 January 2001

Frossard, Jean Louis, Catherine M Pastor, and An- toine Hadengue.Effect of hyperthermia on NF-␬B binding activity in cerulein-induced acute pancreatitis.Am J Physiol Gastrointest Liver Physiol 280: G1157–G1162, 2001.—

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

EXPOSURE OF PROKARIOTICand 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 subse- quent but lethal exposure to hyperthermia. Addition- ally, 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). Addition- ally, 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.

Address for reprint requests and other correspondence: J. L.

Frossard, Division of Gastroenterology, Geneva Univ. Hospital, 1211 Geneva 14, Switzerland (E-mail: [email protected]).

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.

http://www.ajpgi.org G1157

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METHODS

Animals. All experiments were performed according to protocols approved by institutional animal care and the vet- erinary office. Male Wistar rats (75–125 g), obtained from BRL, were bred and housed in standard cages in a thermo- stated (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␮g/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).

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 I␬B proteins.To detect HSP70 protein, Western blot was performed according to the method of Laemmli (16a) using minigels (Bio-Rad, Zurich, Switzerland). Proteins (10␮g) were loaded in each lane. After gel electrophoresis, proteins were transferred to nitrocellulose membrane accord-

ing 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 ␮g 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 Bio- technology), 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␮g/ml), leupeptin (5␮g/ml), and aprotinin (5␮g/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 vor- texed 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␮g) 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 ␮g 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 complemen- tary oligonucleotide with a 5⬘-G overhang and end-labeled using Klenow DNA polymerase I. Samples were electropho- resed at room temperature in 0.5⫻TBE buffer (1⫻TBE⫽ 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA) on

Fig. 1. Study protocol is shown. Rats were immersed in hot water (42°C) for 20 min and studied 12 h after hyperthermia at the peak of heat shock protein 70 (HSP70) expression within the pancreas. Rats were injected twice at a 1-h interval with a supramaximally stimulating dose of cerulein and killed 2 and 4 h after the second injection of cerulein, unless otherwise cited.

G1158 HYPERTHERMIA AND CERULEIN-INDUCED PANCREATITIS

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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 commer- cially 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 (14,000gfor 5 min at 4°C), and quantitating TNF-␣ in the resulting supernatant. Results were expressed as picograms per mi- crogram 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␮m). After staining with hematoxylin and eosin, the sections were examined by an experienced mor- phologist 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 determina- tions. The significance of changes was evaluated using Stu- dent’st-test when data included two groups or ANOVA when three or more groups were compared.

RESULTS

Hyperthermia induced intrapancreatic HSP70 pro- tein 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).

Effects of hyperthermia on severity of cerulein-in- duced 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, concomi-

tantly increased intrapancreatic MPO activity. In con- trast, 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.

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/␮g DNA at 3 h; 6.44 ⫾ 0.48 pg/␮g DNA at 5 h) than in the presence of hyperthermia (1.42⫾0.2 pg/␮g DNA at 3 h; 2.56⫾0.26 pg/␮g DNA at 5 h) (P⬍0.01).

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

Fig. 2. Effects of hyperthermia on pancreatic HSP70 expression overtime. Western blot for HSP 70 expression was performed using monoclonal mouse anti-HSP70 antibody. The Western blot is representative of 3 experiments. C, control.

Fig. 3. Effects of hyperthermia on the severity of cerulein-induced pancreatitis. Rats were given 2 injections of cerulein at a 1-h interval (10␮g/kg ip) or saline and killed 3 and 5 h after the start of cerulein.

Serum amylase activity, pancreas water content (top), myeloperoxidase (MPO) activity, and acinar cell necrosis (bottom) were quantitated as described inMETHODS. Results are expressed as %maximal response obtained at 3 and 5 h. Filled bars, control rats; open bars, control rats exposed to hyperthermia; hatched bars, cerulein-injected rats in the absence of hyperthermia; crosshatched bars, cerulein injected in animals previously exposed to hyperthermia. Results are means⫾SE forⱖ12 animals in each group. Vertical bars denote SE values and the absence of such bars indicates that the SE was too small to be illustrated. *P⬍0.01 vs. cerulein-injected rats in the absence of hyperthermia.

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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).

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. How- ever, 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.

Because previous studies showed that the heat shock response inhibits NF-␬B nuclear translocation by sta-

Fig. 6. Effect of hyperthermia on nuclear factor-␬B (NF-␬B) activity during cerulein-induced pancreatitis. After cerulein injections (10

␮g/kg ip injected twice), rats were killed at 30, 45, 60, 90, and 120 min. A: electrophoretic mobility shift assay performed on nuclear extracts isolated from normal and cerulein-treated (Cer) rat pancreas in the presence or absence of hyperthermia (Heat). B: the intensities of NF-␬B bands that were densitometrically quantified.

Results are mean⫾SE; 5 animals were subjected to hyperthermia before cerulein injection (^F) and 5 animals had no hyperthermia (^Œ).

*P⬍0.05 vs. cerulein-injected rats in the absence of hyperthermia.

Fig. 4. Effect of hyperthermia on the time-dependent increase of tumor necrosis factor-␣(TNF-␣) level during cerulein-induced acute pancreatitis : After cerulein injections (10␮g/kg ip injected twice), rats were killed every hour for 5 h, and pancreatic tissue was rapidly removed and assayed for TNF-␣. Results are mean⫾SE; vertical bars denote SE values and the absence of such bars indicates that the SE was too small to be illustrated. Eight animals were subjected to hyperthermia (^F) and eight animals were not (^Œ). *P⬍0.05 vs.

cerulein-injected rats in the absence of hyperthermia.

Fig. 5. Effect of hyperthermia on intercellular adhesion molecule-1 (ICAM-1) expression in the pancreas. After cerulein injections (10

␮g/kg ip injected twice), pancreatic tissue was immediately har- vested and kept at⫺80°C for ICAM-1 expression.A: a representative Western blot using mouse anti-ICAM-1 monoclonal antibody. C, saline-treated rats; C*, saline and hyperthermia; Cer, cerulein; Cer*, hyperthermia and cerulein.B: the intensities of ICAM-1 bands that were densitometrically quantified (4 different experiments). Results are means⫾SE; 4 animals were subjected to hyperthermia before cerulein injection (^F) and 4 animals had no hyperthermia (^Œ). *P⬍ 0.02 vs. cerulein-injected rats in the absence of hyperthermia.

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bilizing the NF-␬B inhibitory protein I␬B-␣ (23), we collected cytoplasmic extracts from rats treated with cerulein in the absence and presence of hyperthermia and analyzed them by Western blot using anti-inhibitory ␬B-␣ (I␬B-␣) antibody (Fig. 7). The cytoplasmic I␬B-␣signal decreased 30 min after cerulein adminis- tration in the absence of hyperthermia (⫺57% compared with pancreas isolated from animals injected with saline), suggesting the nuclear translocation of NF-␬B, whereas I␬B-␣ signal remained in the cytoplasm until 60 min in the presence of hyperthermia, suggesting the preservation of the NF-␬B-I␬B-␣ complex at this time (Fig. 7).

DISCUSSION

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 hyperther-

mia. 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 mi- crovascular 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 re- cruitment 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 disap- peared 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). Sev- eral 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 interfer- ing 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 in-

Fig. 7. Effect of hyperthermia on inhibitory␬B-␣(I␬B-␣) degradation during cerulein induced-acute pancreatitis. After cerulein injections, rats were killed every 30 min during 2 h. For I␬B-␣measurement, cytosolic protein extracts were analyzed using the Western blot technique. Each Western blot is representative of 5 experiments.

Results are mean⫾ SE. Optical densities were as follows: 0 min, Cer⫽1.74⫾0.22 and Heat⫹Cer⫽1.62⫾0.18; 30 min, Cer⫽ 0.98⫾0.15 and Heat⫹Cer⫽1.09⫾0.11. 45 min, Cer⫽0.17⫾0.09 and Heat⫹Cer⫽0.86⫾0.1; 60 min, Cer⫽0.12⫾0.05 and Heat⫹ Cer⫽1.18⫾0.13; and 90 min, Cer⫽0.44⫾0.15 and Heat⫹Cer⫽ 0.13⫾0.05.

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volved in multiple aspects of cell regulation (8). The molecular protective mechanism of hyperthermia is not fully understood; further study is needed to com- prehend 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 sug- gests that the heat shock pathway is able to simulta- neously switch on cytoprotective genes and downregu- late genes encoding for proinflammatory cytokines such as TNF-␣.

This work was supported by a grant from the Swiss National Science Foundation (32.63618.00).

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