Lipopolysaccharide Directly Affects Pancreatic Acinar Cells

Lipopolysaccharide Directly AffectsPancreatic Acinar Cells

Implications on Acute Pancreatitis Pathophysiology

MARIA I. VACCARO, PhD, EZEQUIEL L. CALVO, MD, ANGELA M. SUBURO, MD,DANIEL O. SORDELLI, PhD, GUSTAVO LANOSA, MD, and JUAN L. IOVANNA, MD

We have explored whether lypopolisaccharide (LPS, endotoxin) induces pancreatic injury onpancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with fourintraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasingdoses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was stronglyinduced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS alsoinduced apoptosis and expression of TNF-a, IL-1b, and IL-8 mRNA in AR4-2J cells. The invivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmicvacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demon-strated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells.The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effectLPS has on the acinar cell.

KEY WORDS: pancreatic acinar cells; acute pancreatitis; lipopolysaccharide; pancreatitis-associated protein; cyto-kines.

Lipopolysaccharide (LPS, endotoxin) of the gram-negative bacteria outer membrane plays a central rolein the pathophysiology of the sepsis syndrome (1).Effects of LPS may lead to organ dysfunction orfailure (2, 3). LPS has been linked to the pathogenesisof acute pancreatitis. Clinically, the degree of endo-toxemia is indicative of severe disease (4). Recently,Wig et al (5) have demonstrated that the presence ofendotoxin in blood and peritoneal fluid correlateswith the severity, systemic complications, and mortal-ity rates of acute pancreatitis. Interestingly, LPS hasbeen found in plasma of patients suffering from se-vere pancreatitis at an early stage of the disease.

Several case reports relate gram-negative infection toacute pancreatitis (6, 7). Changes resembling acutepancreatitis also were described after administrationof LPS to several animal species (8–11). However,little is known about the role of LPS in the inductionof the acute disease of the pancreas. Moreover, it isnot clear whether LPS exerts damage to the pancreasdirectly on the acinar cell. Finally, LPS could affectthe pancreas by producing activation of cytokines.Although the ability of LPS to induce the release ofcytokines by activated monocytes is well known (12),it is not certain whether LPS is able to induce theexpression of cytokines in acinar cells.

In a previous study (11) we showed that chronic S.typhi LPS infusion in rats induced pancreatic morpho-logical and functional features compatible with acutepancreatitis. In the current study we investigatedwhether LPS can exert a direct effect on the pancreasacinar cell. We used the rat pancreatic cell line AR4-2J, which proved to be a suitable model to study the

Manuscript received April 15, 1999; accepted August 30, 1999.From the Universidad de Buenos Aires and Universidad Austral,

Buenos Aires, Argentina; Sherbrooke University, Quebec, Canada;and INSERM U315, Marseille, France.

Supported by Fundacion Antorchas, Universidad de BuenosAires and ANPCyT, Argentina.

Address for reprint requests: Marıa Ines Vaccaro, Universidadde Buenos Aires, Puan 381 p3, 1406 Buenos Aires, Argentina.

Digestive Diseases and Sciences, Vol. 45, No. 5 (May 2000), pp. 915–926

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in vitro response of acinar cells to several inflamma-tory agents (13, 14). The expression of pancreatitis-associated protein (PAP) was assayed as a marker ofacute pancreatitis (15). We show that PAP gene ex-pression is activated, whereas amylase expression isinhibited, in response to LPS in AR4-2J cells. In thesecells, LPS also induces apoptosis studied by monitor-ing DNA fragmentation. We also demonstrate thatLPS is able to activate the gene expression of proin-flammatory cytokines by AR4-2J cells. Finally, theeffect of LPS on acinar cells is shown in vivo. Intra-peritoneal injection of LPS in rats produces numer-ous cytoplasmic vacuoles, loss of cellular polarity andfocal acinar cell apoptosis, as well as significant ex-pression of PAP in pancreatic tissue. The role of LPSin the pathophysiology of acute pancreatitis may bepartly due to the effect LPS has on the acinar cell.

MATERIALS AND METHODS

Cell Cultures. The AR4-2J pancreatic acinar cell line wasobtained from Dr. A. Estival (INSERM U151, Toulouse,France) and cells were used after 42–49 passages. Cellswere routinely cultured at 37°C in a 5% CO2, 95% airatmosphere in Dulbecco’s modified Eagle’s medium(Gibco-BRL) containing 10% (v/v) fetal calf serum (Gibco-BRL), 4 mM L-glutamine, 50 units/ml of penicillin and 50mg/ml streptomycin. When cells reached 80–90% conflu-ence, they were dissociated with 0.05% trypsin and 0.02%EDTA in Puck’s saline A and replated onto 100-mm Petridishes.

Animals. Male Wistar rats weighing 180–200 g wereused. The animals were maintained on a 12L:12D cycle andallowed free access to standard rodent chow and tap water.This investigation was performed according to the Guidefor the Care and Use of Laboratory Animals [DHEWPublication No. (NIH) 85-23, Revised 1985, Office of Sci-

Fig 1. PAP mRNA induction by LPS in AR4-2J cells. Cells were incubated at 37°C in a 5%CO2, 95% air atmosphere in Dulbecco’s modified Eagle’s medium and exposed to 10 mg/mlof LPS during the indicated period. The total RNA was isolated, submitted to electrophore-sis (20 mg/lane) through a formaldehyde–agarose gel, transferred to a nylon membrane, andhybridized to 32P-labeled cDNA for PAP, amylase, and rRNA 18S.

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916 Digestive Diseases and Sciences, Vol. 45, No. 5 (May 2000)

ence and Health Reports, DRR/NIH, Bethesda, Maryland20892)].

Experimental Protocols. AR4-2J cells were incubated inculture medium and exposed to different LPS doses orsterile nonpyrogenic saline during the indicated period.LPS (Salmonella typhi, endotoxin SA . 500,000 units/mg)was purchased from Sigma Chemical Co. (St. Louis, Mis-souri). After the different treatments, approximately 1.2 3107 cells were lysed for RNA extraction (16). For measure-ment of DNA fragmentation 4 3 104 AR4-2J cells weretreated with LPS (10 mg/ml) for periods of different length.

Each experiment was performed in triplicate. Animals werefasted 16 hr before the experiments. Rats were injectedintraperitoneally four times with 10 mg LPS/kg body weightat 1-hr intervals. Control rats were injected intraperitone-ally with an equal volume of sterile nonpyrogenic salinefollowing the same injection schedule. All the experimentswere started between 8:00 and 9:00 AM. Animals (8 rats/group) were killed by decapitation at different time pointsafter LPS injection. Blood was collected and allowed to clot,and serum amylase was assayed using a standard method(17). The pancreas was removed and immediately trimmed

Fig 2. LPS dose–response curve. AR4-2J cells were incubated at 37°C in a 5% CO2, 95%air atmosphere in Dulbecco’s modified Eagle’s medium and exposed to increasingconcentration of LPS for 18 hr. The total RNA was isolated, submitted to electrophore-sis (20 mg/lane) through a formaldehyde–agarose gel, transferred to a nylon membrane,and hybridized to 32P-labeled cDNA for PAP and RNA 18S: (A) northern analysis; (B)quantitative densytometric data of PAP mRNA/rRNA 18S. Data are expressed as themean 6 SEM of three experiments.

LPS AND ACUTE PANCREATITIS

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free of fat in cold phosphate-buffered saline (PBS). ForWestern blot analysis, one piece from each pancreas wasfrozen under liquid nitrogen and stored at 280°C. Anotherpiece was minced and fixed for electron microscopy. Theremainder of each gland was processed immediately forRNA extraction.

Visualization and Quantification of Genomic DNA Frag-mentation. Following cell treatment with LPS, attached andfloating cells were collected by centrifugation, lysed byresuspension in lysis buffer containing 10 mM Tris HCl, pH7.4, 10 mM EDTA, 0.1% SDS, 20 mg/ml of pancreaticRNase, and 100 mg/ml proteinase K and incubated at 50°Cfor 6 hr. DNA was purified by two phenol–chloroformextractions (1:1, v/v), precipitated with 1:4 (v/v) 10 M so-dium acetate, and collected by centrifugation at 15,000g for15 min at 4°C. DNA was precipitated with ethanol andresuspended. DNA fragments were separated electro-phoretically on 1.5% agarose gel containing 0.5 mg/mlethidium bromide in 0.53 TBE buffer, and visualized underUV light. Quantitation of DNA fragmentation was mea-sured using a photometric enzyme-linked immunosorbentassay (ELISA) for the detection of BrdU-labeled DNAfragments (Boehringer Mannheim). Briefly, AR4-2J cellswere batch-labeled with 10 mg/ml BrdU overnight andplated in 96-well microtiter plates. Twenty-four hours later,they were treated with LPS for different incubation periods.Culture supernatants were pipetted in microtiter platesprecoated with a monoclonal anti-DNA antibody that bindsto DNA strand breaks. BrdU-labeled DNA fragments weredetected after incubation with anti-BrdU-peroxidase conju-gates by photometric measurement on an ELISA platereader at 450 nm (reference wavelength 690 nm).

Probe Preparation. The PAP complementary DNA(cDNA) probe was a 780-bp fragment with complete codantsequence (GeneBank accession number M55149) (18). Theamylase cDNA probe was a 1100-bp fragment from clonepCXP100 (15). The TNF-a cDNA probe was a 1100-bpEcoRI insert from the pBR322-derived vector (19). TheIL-1b cDNA probe was made with a 1350-bp EcoRI insertfrom pBR322-derived vector (20). The IL-8 cDNA probewas made with a 478-bp EcoRI insert from pUC19 vector.These three vectors were generously provided by Genen-tech (San Francisco, California). The cDNA probes werelabeled with [32P]dCTP by random priming using a com-mercial kit (Megaprime DNA Labelling System, Amer-sham).

Northern Blot Analysis. Twenty micrograms of totalRNA (quantitated by measurement of absorbance at 260nm) were size-fractionated on a 1% agarose gel containing2.2 M formaldehyde and transferred to nylon membranes(Nytran Plus, Schleicher and Schuell). Membranes wereprehybridized at 42°C for 4–6 hr in 53 SSC, 50 mMHEPES (pH 6.8), 1% SDS, 53 Denhardt’s, 2 mM EDTA,50% formamide, and ssDNA (200 mg/ml). Hybridizationwas performed at 42°C over 16–20 hr in the solution indi-cated above containing the [32P]dCTP cDNA probe (1–2 3106 cpm/ml). After hybridization, filters were washed with0.13 SSC, 0.1% SDS twice, 30 min at 56°C, and exposed toa Kodak X-Omat AR film, with an intensifying screen at280°C for variable periods of time. A 18S ribosomal[32P]dCTP-labeled cDNA probe was used as a control toevaluate RNA loading and transfer. Radioautograms were

evaluated by means of a laser densitometer (BioRad Imag-ing densitometer, model GS-670, BioRad LaboratoriesLtd.).

Western Blot Analysis. Frozen pancreases were homog-enized in 50 mM Tris HCl (pH 7.5) with 100 mM NaCl, 20mg/ml aprotinin, 20 mg/ml leupeptin, and 0.5 mM PMSF.

Fig 3. Apoptosis in AR4-2J cells treated with LPS. (A) Ethidiumbromide-stained agarose gel containing genomic DNA isolatedfrom AR4-2J cells incubated at 37°C in a 5% CO2, 95% airatmosphere in Dulbecco’s modified Eagle’s medium and exposedto 10 mg/ml of LPS for 24 hr. (B) AR4-2J cells were prelabeled with5-bromo-29-deoxyuridine for 18 hr. Twenty-four hours leter, cellswere treated with 10 mg/ml LPS and the 5-bromo-29-deoxyuridine-labeled DNA fragments were assayed in the supernatant at theindicated times using an ELISA test. Data are expressed as themean 6 SEM of three experiments.

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The primary anti-rat PAP antibody was a rabbit polyclonalantibody (21). After incubation, the filters were washed andincubated further for 1 hr at room temperature with horse-radish peroxidase-linked F(ab)2 fragment anti-rabbit sec-ondary antibody (1:4.000, Amersham). The filters were thenwashed and exposed to the Super Signal substrate detectionsystem (Pierce).

Histological Analysis. Tissues were fixed in 1% glutaral-dehyde in 0.1 M phosphate buffer and postfixed in osmiumtetroxide in the same buffer. After immersion in uranylacetate, the samples were dehydrated in graded concentra-tions of ethanol and cleared with acetone. They were em-bedded in Embed 812 (Electron Microscopy Scientific).Sections 1 mm thick were stained with 1% methylene blue inborax whereas ultrathin sections were stained with leadcitrate and uranyl acetate. Grids were examined under a

Zeiss electronmicroscope at the laboratory of the Funda-cion Oftalmologica Argentina.

RESULTS

LPS Directly Damaged AR4-2J Cells. In order toknow whether LPS is able to affect the pancreas cellsdirectly, the effect of LPS on the expression of PAPand amylase mRNA were examined in the rat acinarcell line AR4-2J. Northern analysis of PAP and amy-lase mRNA in a time-dependent treatment of AR4-2Jcells with LPS is shown in Figure 1. Exposure ofAR4-2J cells to 10 mg/ml LPS determined a stronginduction of PAP mRNA expression after 18 hr of

Fig 4. Cytokine mRNA induction by LPS in AR4-2J cells. Cells were incubated at 37°Cin a 5% CO2, 95% air atmosphere in Dulbecco’s modified Eagle’s medium and exposedto 10 mg/ml of LPS during the indicated period. The total RNA was isolated, submittedto electrophoresis (20 mg/lane) through a formaldehyde-agarose gel, transferred to anylon membrane, and hybridized to 32P-labeled cDNA for TNF-a, IL-1b, IL-8, andrRNA 18S.



Fig 5. This series of light micrographs from 1-mm thin plastic sections illustrates the structural charac-teristics of acinar cells at 0, 12, 24, 36, 48, and 72 hr (A, B, C, D, E, and F, respectively) after administrationof LPS. Wistar male rats were injected intraperitoneally four times with 10 mg LPS/kg body weight at 1-hrintervals. At different time points after LPS injection, the pancreas was fixed in 1% glutaraldehyde in 0.1M phosphate buffer and postfixed in osmium tetroxide in the same buffer. Samples were embedded inEmbed 812, and 1-mm thick sections were stained with 1% methylene blue in borax. Micrographs in A andB were obtained with Nomarski optics. (A) Normal features of pancreatic exocrine cells are observed ina control rat. Notice the polarized organization of the acini, with a central lumen (l) surrounded bynumerous secretory granules and basal nuclei. The diameters of secretory granule profiles are quite similarwithin each cell. Most nuclei have a moderately stained nucleoplasm and their heterochromatin isdistributed in a central clump and several smaller peripheral granules. (B) Tissue fixed 12 hr after LPSadministration. In these acinar cells, secretory granule profiles have a larger diameter than in controlsamples. This Nomarski optics micrograph illustrates the appearance of cytoplasmic vacuoles. Alterationof nuclear heterochromatin is evident in several nuclei (arrowheads), where a large central heterochro-matic clump is surrounded by a pale nucleoplasm. (d), nuclei of a ductal cell. (C) These damaged acini

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incubation, while the expression of amylase mRNAwas reduced. The membranes were reprobed withrRNA 18S cDNA as an internal control to ensureequal loading of RNA in each lane. Figure 2 showsthe levels of PAP mRNA in AR4-2J cells after 18 hrof exposure to increasing concentrations of LPS from1023 to 102 mg/ml. Levels of PAP mRNA were in-creased after treatment with LPS at concentrations of1022 mg/ml, with maximum expression at 10 mg/ml.The quantitative densitometry data from three exper-iments are also shown in Figure 2.

The effect of LPS on DNA fragmentation wasinvestigated in AR4-2J cells after 24 hr of incubationwith 10 mg/ml of LPS in order to determine whetherLPS treatment could damage pancreatic acinar cells.Genomic DNA was extracted and analyzed by elec-trophoresis as described in Materials and Methods.As shown in Figure 3, DNA fragmentation was ob-served in AR4-2J cells treated with LPS. Similarresults were obtained when DNA fragmentation wasmeasured using an ELISA test in a time-dependenttreatment with 10 mg/ml LPS (Figure 3).

LPS Induced Expression of Proinflammatory Cy-tokines in AR4-2J Cells. For the purpose of testingwhether AR4-2J cells expressed cytokines in responseto LPS, we investigated the expression of TNF-a,IL-1b, and IL-8 mRNA in control preparations andunder LPS treatment. Northern analyses of cytokinemRNA in a time-dependent treatment of AR4-2Jcells with 10 mg/ml LPS are shown in Figure 4. Con-trol cells did not exhibit expression of the testedcytokines. Exposure of AR4-2J cells to 10 mg/ml LPSinduced TNF-a mRNA transcripts with maximal ex-pression after a 12-hr incubation period. LPS alsoinduced the expression of IL-1b and IL-8 mRNAtranscripts that were maximal in 18 hr. The mem-branes were reprobed with the rRNA 18S cDNA ascontrol.

In Vivo Effect of LPS on Rat Pancreas Acinar Cells.Repeated intraperitoneal injection of LPS in a non-lethal dose induced a significant increase in serumamylase activity (3512 6 275 vs 1533 6 87 units/liter,N 5 8, P , 0.001) 24 hr after initiation of LPS

treatment. There were no macroscopic signs of mes-enteric fat necrosis. Most of the animals survived thecourse of LPS injections.

The structure of the exocrine pancreatic tissue wasanalyzed in specimens fixed 6, 12, 24, 36, 48, and 72 hrafter administration of LPS, under both light andelectron microscopy. Structures are shown in Figures5 and 6, respectively. Initial changes in the numberand heterogeneity of zymogen granules were evidentafter 6 hr, but became much more marked 12 hr afterthe first LPS injection. At this time (Figure 5B), manynuclei showed central heterochromatic clumps sur-rounded by a very pale nucleoplasm. Electron micros-copy (Figure 6B and C) demonstrated wide dilata-tions of the endoplasmic reticulum (ER) cisterns, aswell as abnormalities in the size and localizationpattern of zymogen granules. Vacuoles containingelectron dense debris suggest the existence of anintense crynophagy. The cytoplasm also containedfibrillar inclusions. Signs of cell damage greatly in-creased 24 hr after LPS administration. There was alarge reduction in the number of secretory granules(Figure 5C), together with numerous vacuoles in thecytoplasm and enlargement of the basolateral space(Figure 6D and E). Electron microscopy also demon-strated pale cells containing very few zymogen gran-ules and a substantial dilatation of the ER (Figure6D). These cells showed complete disruption of theapical region, with large irregular processes invadingthe lumen. In spite of the existence of apical junc-tional complexes, the basolateral space was widelyenlarged and secretory granules were closely attachedto the basolateral plasma membrane (Figure 6E).Furthermore, cells also showed severe nuclear alter-ations. Many nuclei exhibited complete disappear-ance of chromatin structure or evidence of nuclearfragmentation (Figure 5C). The complete disappear-ance of nuclear structure was accompanied by thetotal loss of secretory granules and a very large dila-tation of the ER (Figure 6F).

In specimens fixed 36 hr after LPS administration(Figure 5D), remaining acini had very few secretorygranules and showed a gross distortion of their nor-

were observed 24 hr after LPS administration. Cell cytoplasm contains numerous vacuoles and dilatations also appear along the basolateralspaces (arrows). Nuclear structure is completely lost and evidence of karyorrhexis appears in some cells (arrowheads). (D) Distortion ofacinar organization is present in this specimen fixed 36 hr after LPS injection. Notice that secretory granules are surrounded by largevacuoles. Alterations of nuclear heterochromatin are similar to those described in micrograph B. A large number of ductal cells (d) is acharacteristic feature of this period. (E) Acinar alterations two days after LPS injection include an almost complete disappearance ofsecretory granules and random positioning of cell nuclei. Most cell boundaries have disappeared. (F) This micrograph illustrates twoneighboring acini showing different degrees of cell damage. In acinus A there is a complete loss of cell boundaries, and both secretorygranules and cell nuclei are randomly dispersed in a large cytoplasmic area. By contrast, acinus B shows apical accumulation of secretorygranules and a slightly vacuolized cytoplasm. A–E, and F: magnification 31100; D: 3 450.



Fig 6. These electron micrographs illustrate different aspects of acinar ultrastructure in control rats (A),12 hr (B and C), and 24 hr (D–F) after LPS injection. Wistar male rats were injected intraperitoneally fourtimes with 10 mg LPS/kg body weight at 1-hr intervals. At different time points after LPS injection, thepancreas was fixed in 1% glutaraldehyde in 0.1 M phosphate buffer and postfixed in osmium tetroxide inthe same buffer. Samples were embedded in Embed 812; ultrathin sections were stained with lead citrateand uranyl acetate. Grids were examined under a Zeiss electronmicroscope. (A) This micrograph from acontrol animal shows the apical accumulation of secretory granules near the acinar lumen (l); n, nucleusof the acinar cell; d, nucleus of a ductal cell. (B) Structural damage is evident in this micrograph obtainedfrom a sample fixed 12 hr after LPS administration. Notice the size of secretory granule profiles, theenlargement of ER cisterns and the presence of fibrillar inclusions (f). The region surrounding thesecretory granule marked with an asterisk is enlarged in the next figure. (C) Notice the dilated ER cisterns,the presence of a heterogeneous population of secretory granules, and large vacuoles (v) containingelectron dense debris suggesting crynophagia. (D) The lumen (l) of this acinus is surrounded by severalcells with an electron dense cytoplasm and another cell with an electron pale cytoplasm (p). The dark cellscontain more secretory granules and less dilated ER cisterns than the pale cell. The integrity of the apical

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mal apicobasal organization. Acinar cells also, weresurrounded by a very large number of ductal-likecells. Almost every acinar cell nucleus exhibited com-pacted heterochromatin and a very pale nucleoplasm;however, the images of karyolysis and karyorrhexisobserved in the 24-hr specimen were seldom found atthis stage. Two and three days after LPS administra-tion (Figure 5E and F), some acini with a nearlynormal structure were mingled with grossly distortedacini, with very few secretory granules scattered overa cytoplasm with many vacuoles and undefined cellboundaries. Cell nuclei showed heterochromatic al-terations and occupied random positions, suggestinga lack of cell polarization. Infiltration of inflammatorycells was not observed at any of the experimentalperiods.

To determine whether LPS treatment induced theexpression of PAP in pancreas, the total soluble pro-tein contents were analyzed by western blot. Anti-PAP antibodies recognized a 15-kDa protein in pan-creatic tissue from rats treated with LPS, whereas noexpression was detected in control rats. The time-course effect of LPS administration on pancreaticPAP expression is shown in Figure 7. PAP was de-tected in the pancreas 6 hr after LPS administration,it was found increased by 36, 48 and 72 hr, thendecreased and became undetectable four days afterLPS administration.

DISCUSSION

The present study demonstrated that the effect ofLPS on exocrine pancreas is mediated by a directaction of the endotoxin on the acinar cell. Experimen-tal studies have described changes resembling acutepancreatitis in several animal models after adminis-tration of LPS. A single injection of endotoxin intothe superior pancreaticoduodenal artery of rabbitsinduced a dose-dependent acute necrotizing pancre-atitis (8). In rats, endotoxemia sustained over 6 hrincreased the levels of pancreatic lipase (10). Also inrats, LPS priming exacerbated acute reflux pancreati-tis (22). Moreover, we have previously demonstratedthat chronic intraperitoneal infusion of LPS induceddecreased protein output and alterations of the pro-tein relative content in rat pancreatic juice (11). Inspite of the fact that several in vivo studies have been

carried out that suggest a close relation between LPSand pancreatitis, it is not clear whether LPS onlybrings about a systemic reaction that causes or wors-ens the disease, or if LPS actually exerts damage tothe pancreas directly at a cellular level. The use of thepancreatic acinar cell line AR4-2J allowed us to es-tablish that LPS can exert a direct action on pancre-atic acinar cells and cause cellular changes compati-ble with pancreatitis. Cell injury is one of the earliestevents in the development of acute pancreatitis. Themolecular events occurring during the course of thedisease involve rapid and precise reorganization ofthe pancreatic cell genetic program and many cellsactivating their apoptotic program (23). Our resultsindicate that LPS can induce phenotypical cellularchanges of pancreatitis in AR4-2J cells, such as de-creased amylase mRNA and expression of PAPmRNA. Recent reports have pointed out that LPSinduces acinar cell apoptosis in animal models. Laineet al showed apoptosis in rat pancreas 24 hr after LPSadministration (9). Even low doses of LPS up-regulated acinar cell apoptosis in rat cerulein pancre-atitis (24). The tissue injury described in these studiescould be secondary to LPS-induced systemic inflam-mation. Nevertheless, we report the ability of LPS toinduce apoptosis acting directly on acinar cells.

In this study, we also demonstrated that LPS is ableto induce gene expression of proinflammatory cyto-kines on pancreatic acinar cells. The ability of LPS toinduce the release of cytokines by activated mono-cytes is well established (12). Classically, TNF-a ex-pression has been associated with the detrimentaleffects of endotoxin exposure (25). Increased TNF-aand IL-1b serum levels have been demonstrated insevere acute pancreatitis (3), and it has been shownthat serum levels of proinflammatory cytokines cor-relate to a high degree with the severity of acutepancreatitis (26, 27). In addition, TNF-a and IL-1bare produced in the pancreas during cerulein acutepancreatitis (28). Although infiltrating leukocytesmight be responsible for TNF-a and IL-1b produc-tion during acute pancreatitis (29), the actual sourceof cytokines during the development of the diseasewas not clearly established. Our experiments showedthat TNF-a, IL-1b, and IL-8 mRNAs were stronglyinduced by LPS in AR4-2J cells. To our knowledge,

surface seems to be preserved in the electron dense cells, but seems to be disrupted in the electron pale cell. Notice the presence of cellprocesses invading the lumen. (E) This micrograph illustrates the dilatation of the basolateral spaces (b). Notice the presence of a junctionalcomplex (arrowhead) and the attachement of secretory granules to the basolateral membrane. (F) This cell nucleus has completely lost itsnormal chromatin structure. Secretory granules have completely disappeared and the ER is enormously dilated. A and B: magnification,36000; C: 312,750; D: 3 9200; E and F: 3 11,000.



there is no previous evidence stating that LPS stimu-lates expression of TNF-a, IL-1b, and IL-8 mRNA bypancreatic acinar cells. The presence of TNF-a inisolated pancreatic acini has been reported from con-trol rats (30). The activation of this cytokine in con-trol acinar cells could be due to the isolation proce-dure undergone by the tissue. In our experiments, onthe contrary, the expression of TNF-a by AR4-2J cellsincubated in control medium was unnoticed; in theseconditions AR4-2J cells did not express PAP either, aspecific pancreatic acute phase protein (13). The in-duction of proinflammatory cytokines mRNA by LPSseems to be part of a reorganization of the geneticprogram of pancreatic cells during the acute responseleading to changes in their phenotype. Moreover,Grady et al (31) reported that gene expression ofmob-1 and mcp-1 (chemokines belonging to the IL-8family) by acinar cells was associated with cerulein-induced acute pancreatitis. Hence, pancreatic acinarcell expression of cytokines during pancreatitis wouldnot be model-dependent.

In vivo studies demonstrated that LPS is able toinduce ultrastructural features characterized as earlycellular events in the pathophysiology of experimentalpancreatitis, such as cytoplasmic vacuolization, loss ofcellular polarity, and focal acinar cell death (32).Ultrastructural characteristics of apoptosis were ob-served after 12 hr of LPS injection. Interestingly,infiltration of inflammatory cells was not detected

during this period. Gukovskaya et al (33) suggestedthat apoptosis correlated with a low level of infiltra-tion. Kimura et al (24), while describing apoptosisafter LPS pretreatment on rats undergoing caeruleinpancreatitis, also noticed that pancreatic infiltrationwas scarce. Hence, the in vivo and in vitro apoptosisfound in our study may be a response to acinar cellinjury induced by LPS (34). Furthermore, 36 hr afterLPS injection, a large number of ductal cells wasobserved. The increase in ductal cells has been usuallyconsidered a consequence of the proliferation of stemcells and/or preexisting ductular cells. However, newevidence about the three-dimensional structure of theexocrine pancreas suggests that, during pancreatitis,acini may undergo redifferentiation, taking on themorphology of ductules (35). In vivo studies alsoshowed expression of PAP in pancreatic tissue fromrats treated with LPS, indicating the induction ofacute pancreatitis. Expression of PAP is maximal36–72 hr after LPS injection. Ultrastructural studiesshowed maximal acinar cell injury 24 hr after LPSadministration; thus PAP expression could resultfrom LPS-induced injury, probably as a part of adefense mechanism of the pancreas against aggres-sion (14).

This study shows that LPS can induce cellularevents resembling acute pancreatitis by a direct effecton pancreatic acinar cells. The mechanism by whichLPS induces those events, however, remains unclear.

Fig 7. Expression of PAP induced by LPS in vivo. Wistar male rats were injected intraperitoneally four times with 10 mg LPS/kgbody weight at 1-hr intervals. At different time points after LPS injection, the pancreas was homogenized in 50 mM Tris HCl(pH 7.5) and western analysis was performed. The primary anti-rat PAP antibody was a rabbit polyclonal antibody. Thesecondary antibody was horseradish peroxidase-linked F(ab)2 fragment anti-rabbit (1:4.000). The filters were exposed to theSuper Signal substrate detection system.

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The autolytic destruction of mitochondrial mem-branes of pancreas exocrine cells reported by Bendeet al (36) or nonspecific hydrolysis of membranephospholipids catalyzed by the early activation ofPLA2 (9) may explain the LPS-induced injury to thepancreatic acinar cell observed in this study. Further-more, LPS may bind to a surface receptor on acinarcells leading to the induction of changes in geneexpression through an unknown transduction path-way and may lead ultimately to pancreatic acinar cellinjury. It has been reported that the lack of CD14 didnot prevent acute pancreatitis induced in mice by acholine-deficient and ethionine-supplemented diet(37), but cell types not expressing CD14 may alsorespond to LPS. It has been demonstrated in therenal tubular epithelial cell line LLC-PK (38) and inisolated renal tubules (39) that LPS stimulates a rapidrise in intracellular Ca21 concentration. Finally, LPScould be absorbed from the bowel into the systemiccirculation because of increased intestinal macromo-lecular permeability. Ryan et al (40) demonstratedthat intestinal permeability correlated with the sever-ity of acute pancreatitis. Chen et al (41) reported thattreatment with a cathartic agent, sennoside, de-creased translocation of endotoxin and reduced mor-tality in the model of acute pancreatitis induced inrats with sodium deoxycholate. The results from ourstudy demonstrated that LPS exerts direct damage topancreatic cells and lend support to the hypothesisthat LPS may be involved in the pathophysiology ofacute pancreatitis leading to severe disease.

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Lipopolysaccharide Directly Affects Pancreatic Acinar Cells