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GASTROENTEROLOGY 2005;129:1251–1267

xtracellular Cleavage of E-Cadherin by Leukocyte Elastaseuring Acute Experimental Pancreatitis in Rats

ULIA MAYERLE,* JÜRGEN SCHNEKENBURGER,‡ BURKHARD KRÜGER,§ JOSEF KELLERMANN,¶

ANUEL RUTHENBÜRGER,* F. ULRICH WEISS,* ANGEL NALLI,‡ WOLFRAM DOMSCHKE,‡ andARKUS M. LERCH*

Department of Gastroenterology, Endocrinology and Nutrition, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald; ‡Department of Medicine, Westfälische Wilhelms-Universität Münster, Münster; §Department of Pathology, Division of Medical Biology, Universität Rostock, Rostock;
nd ¶Max-Planck-Institut of Biochemistry, Martinsried, Germany
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ackground & Aims: Cadherins play an important role inell-cell contact formation at adherens junctions. Duringhe course of acute pancreatitis, adherens junctions arenown to dissociate—a requirement for the interstitialccumulation of fluid and inflammatory cells—but thenderlying mechanism is unknown. Methods: Acute pan-reatitis was induced in rats by supramaximal ceruleinnfusion. The pancreas and lungs were either homoge-ized for protein analysis or fixed for morphology. Pro-ein sequencing was used to identify proteolytic cleav-ge sites and freshly prepared acini for ex vivo studiesith recombinant proteases. Results were confirmed inivo by treating experimental pancreatitis animals withpecific protease inhibitors. Results: A 15-kilodaltonmaller variant of E-cadherin was detected in the pan-reas within 60 minutes of pancreatitis, was found to behe product of E-cadherin cleavage at amino acid 394 inhe extracellular domain that controls cell-contact for-ation, and was consistent with E-cadherin cleavage by

eukocyte elastase. Employing cell culture and ex vivocini leukocyte elastase was confirmed to cleave E-adherin at the identified position, followed by dissoci-tion of cell contacts and the internalization of cleaved-cadherin to the cytosol. Inhibition of leukocyte elas-ase in vivo prevented E-cadherin cleavage during pan-reatitis and reduced leukocyte transmigration into theancreas. Conclusions: These data provide evidencehat polymorphonuclear leukocyte elastase is involvedn, and required for, the dissociation of cell-cell contactst adherens junctions, the extracellular cleavage of E-adherin, and, ultimately, the transmigration of leuko-ytes into the epithelial tissue during the initial phase ofxperimental pancreatitis.

adherins comprise a family of transmembrane pro-teins that are located at adherens junctions (for

eview see Troyanovsky1) and display calcium-bindingotifs in their extracellular domain, which are essential

or homophilic cell adhesion. In epithelial organs, E-
adherin is the most abundant regulator of adherens
unctions, and its extracellular domain is composed of 5ubunits, EC-1 through EC-5, and the homophilic ad-esion activity of this molecule has been mapped to themino terminal EC-1 domain.2 X-ray characterization ofhis domain revealed a 7-stranded (A-G) �-sandwichtructure,3 and adhesive interaction seems to be driveny the �-sandwich topology. This region does not, intself, participate in Ca2� binding but includes an HAVequence (in single letter code for amino acids) thatediates adhesive interactions.4,5 The intracellular do-ains serve as highly conserved linkers to the cytoskel-

ton via connecting �- and �-catenins.6 These intracel-ularly located proteins are essential for cell-celldhesion, and mutations in either the E-cadherin-bind-ng site for �- and �-catenin or in the catenins them-elves will disrupt cell contacts even in the presence of anntact extracellular E-cadherin domain.7 Although theres extensive knowledge about the role of E-cadherinutations, E-cadherin down-regulation, processing, and

ubcellular relocalization during tumor development oralignant growth,8 there is only limited information on

he role of E-cadherin in inflammation.Epithelial cell-cell contacts at adherens junctions form

selective barrier and are involved in the active transportf fluids, ions, and small molecules. During inflamma-ory disorders, cell-cell contacts frequently dissolve,hich permits an unregulated movement of fluids and

lectrolytes into the interstitial space, resulting in tissuedema. In a model system for an inflammatory disorderexperimental pancreatitis), we have shown that thisdema formation is associated with a dissociation ofdherens junctions between epithelial acinar cells and a

Abbreviations used in this paper: CCK, cholecystokinin; PMN, poly-orphonuclear.

© 2005 by the American Gastroenterological Association0016-5085/05/$30.00

doi:10.1053/j.gastro.2005.08.002

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1252 MAYERLE ET AL GASTROENTEROLOGY Vol. 129, No. 4

isappearance of E-cadherin from their basolateral mem-rane.9

In the present study, we characterized the early eventsnvolved in cell-cell contact dissociation, edema forma-ion, and cell damage during pancreatitis and focussed onhe mechanism that causes the disappearance of E-cad-erin from the cell surface. In theory, the process in-olved in the dissociation of E-cadherin-mediated cellontacts should include cytotoxic or proteolytic agentshat are released by either inflammatory cells10–13 orriginate from the pancreatic acinar cell itself.14–16 Prioro the start of our project, the second hypothesis ap-eared more likely because (1) inflammatory cells werehought to play a role only in the later disease course butot the initial events of experimental pancreatitis, and2) the early phase of pancreatitis is known to be associ-ted with the generation of large amounts of cytotoxicompounds such as free oxygen radicals and an extensivectivation of proteolytic digestive enzymes.17–20 We wereurprised to learn from our experiments that E-cadherins cleaved very early in the disease course (within the firstour) by polymorphonuclear (PMN) granulocyte elas-ase, whereas pancreatic proteases are neither requiredor involved in this process. Moreover, inhibition ofMN elastase not only prevented E-cadherin cleavagend cell-cell contact dissociation but also pancreatic in-ammation and leukocyte infiltration during pancreati-is. These findings provide the first direct evidence for aole of inflammatory cells in the initial disease phase ofancreatitis and designates them a prospective target foruture treatment strategies.

Materials and Methods

Materials

Cerulein was obtained from Pharmacia, Freiburg, Ger-any. Collagenase from Clostridium histolyticum (EC.3.4.24.3) was

rom SERVA (lot No. 14007, Heidelberg, Germany; collagenasectivity, 1.50 PZ U/mg). Human neutrophil elastase was pur-hased from Calbiochem (San Diego, CA; catalog No. 324681;rotein concentration, �20 U/mg protein specific activity; in 50mol/L Na-acetate, pH 5.5, and 200 mmol/L NaCl; purity95%). Bovine pancreatic trypsin, human myeloperoxidase, por-

ine pancreatic elastase, bovine pancreatic chymotrypsin, andovine �-amylase were obtained from Calbiochem (Schwalbach,ermany). The substrates rhodamine 110 (R110)-(CBZ-Ile-Pro-rg)2 and R110-(CBZ-Ala4)2 were purchased from Molecularrobes (Eugene, OR). The substrate 7-amino-4-methylcoumarin

AMC)-(Suc-Ala2-Pro-Phe) was obtained from Bachem (Heidel-erg, Gemany). The amylase quantification kit “Amyl” is com-ercially available from Roche (Ingelheim, Germany). Elastase

nhibitor II was from Calbiochem (catalog No. 324744; San
iego, CA). The biologically active phosphorylated cholecystoki- c
in (CCK) octapeptide [Tyr(SO3H)27]-cholecystokinin fragmentas obtained from Sigma (Taufkirchen, Germany, catalog No.175). For the detection of E-cadherin, 2 different antibodies weresed: monoclonal anti-E-cadherin clone 36 directed against the-terminus (catalog No. 20820; Transduction Laboratories, Saniego, CA) and polyclonal rabbit anti-E-cadherin H108 directed

gainst the N-terminus (catalog No. SC-7870; Santa Cruz, CA).or the detection of human neutrophil elastase, mouse monoclo-al antibody clone AHN-10 (catalog No. MAB1056, lot58CCD; Chemicon International, Temecula, CA) was used. Flu-rescein isothiocyanate (FITC)-conjugated mouse anti-rat CD45leukocyte common antigen) monoclonal antibody was used toabel and quantitate inflammatory cells in paraffin sections duringifferent intervals of pancreatitis (clone OX-1; BD Pharmingen,eidelberg, Germany).All other chemicals were of highest purity and were ob-

ained either from Sigma-Aldrich (Eppelheim, Germany),erck (Darmstadt, Germany), Amersham Pharmacia Biotech

Buckinghamshire, United Kingdom), or Bio-Rad (Hercules,A). Animals were bred at Charles River Breeding Laborato-

ies (Sulzbach, Germany). All animal experiments were con-ucted according to the guidelines of the local animal use andare committee. PaTu-8988-S cells were purchased fromSZM (Braunschweig, Germany).Induction of acute, cerulein-induced pancreatitis, maleistar rats (140–250 g) were anesthetized with pentobarbital

0 mg/kg. A cannula was placed into the jugular vein, and thenimals were infused with supramaximal concentrations oferulein (10 �g/kg per hour) for up to 48 hours or treated witheutrophil elastase inhibitor II at a concentration of 50 �mol/LCalbiochem, San Diego, CA) for 2 hours or a mixture oferulein (10 �g/kg per hour) and neutrophil elastase inhibitor50 �mol/L). Saline-infused animals served as controls. Afterxsanguination under ether anesthesia, the pancreas was rap-dly removed and trimmed of fat, and tissue blocks werembedded in OCT (Tissue Tek, Sakura Finetek, Zoeterwoude,he Netherlands) for cryosections or fixed in 5% formaldehyde

or electron microscopy (EM) cryolabelling or embedding inaraffin. The main part of the pancreas was frozen in liquiditrogen and stored at �80°C for later protein analysis andetection of enzymatic activity.21 Pancreatic tissue was ho-ogenized with a Dounce S glass homogenizer in iced Triton-100 lysis buffer containing protease inhibitors and subse-uently immunoprecipitated and immunoblotted (Braun-Mel-ungen, Melsungen, Germany). Iced Triton X-100 lysis bufferontained protease inhibitors (1 mL/mg tissue, 10 �g/mLprotinin, 10 �g/mL leupeptin, 0.01 mol/L sodium pyrophos-hate, 0.1 mol/L sodium fluoride, 1 mmol/L hydrogen perox-de, 1 mmol/L L-phenylmethylsulfonylfluoride [PMSF], and.02% soybean-trypsin inhibitor). Protein concentration wasetermined by a modified Bradford-assay (Bio-Rad, Unter-aching, Germany), and equal amounts of protein were used inubsequent experiments. DNA content of homogenates wasetermined with propidium iodine using a fluorescence readerexcitation 350 nm/emission 630 nm) and used to standardize
ell homogenates to comparable cell numbers and corrected for

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October 2005 E-CADHERIN SHEDDING IN ACUTE PANCREATITIS 1253

nterstitial serum proteins in pancreatitis animals. For immu-oprecipitation, a mixture of protein A and G-Sepharose (Am-rsham Pharmacia Biotech, Freiburg, Germany) was preincu-ated with antibody in 20 mmol/L HEPES, pH 7.4. Lysatesere precleared with rat nonimmune serum, added to the

oupled antibody, and incubated for 1 hour at 4°C on a rotorheel. Precipitates were washed with HNTG (washing buffer

ontaining HEPES 50 mmol/L, NaCl 150 mmol/L, Triton-X00 0.1%, Glycerin 10%) and boiled for 5 minutes in 2X SDSample buffer. SDS polyacrylamide gel electrophoresis waserformed in a discontinuous buffer system, and gels werelotted on nitrocellulose membranes (Hybond C, Amershamharmacia Biotech). After overnight blocking in NET-(0.2gelatine; washing buffer containing NaCl 1.5 M, EDTA

.05 M, Tris-HCl 0.5 M [pH 7.5]), immunoblot analysis waserformed, followed by enhanced chemoluminescence detec-ion (Amersham Pharmacia Biotech) using horseradish perox-dase coupled sheep anti-mouse IgG (Amersham Pharmacia) oroat anti-rabbit IgG (Amersham, Pharmacia Biotech).22,23

ensitometric analysis of the E-cadherin cleavage product inigure 4 was undertaken using Western blots from 3 differentxperiments, and error bars represent mean values in percent-ge � SEM.

Protein Sequencing

E-cadherin was immunoprecipitated using the mono-lonal clone 36 antibody from Transduction Laboratories asescribed above. Samples were subjected to SDS-PAGE. Gelsere equilibrated in 10 mmol/L CHAPS, pH 11, and meth-

nol (10% vol/vol) for 30 minutes. Semidry transfer ontoolyvinylidene difluoride membranes (PVDF) (catalog No.62-0812, 0.2 �m, lot 149548A, Bio-Rad, Hercules, CA) wasarried out at 1 mA/cm2 constant current for 3 hours at 15°Cn 10 mmol/L CHAPS, pH 11, and methanol (10% vol/vol).VDF membranes were stained with Coomassie brilliant blue-250 stain (Bio-Rad) 0.1% wt/vol and 50% vol/vol methanol

or 15 minutes. Destaining was done in a solution containing0% vol/vol methanol. Protein containing polyvinylidene di-uoride membrane sections were excised and sequenced on aulsed liquid phase sequencer (Applied Biosystems) accordingo the manufacturer’s instructions.24

Assessment of Acinar Cell-CellContact Dissociation

Pancreatic acini were prepared by collagenase digestionCollagenase Serva, Heidelberg, Germany) as previously de-cribed.15 Acini were then washed and centrifuged at 50g for 1inute in DMEM medium containing 0.2% BSA. Incubation

uffer consisted of DMEM, 0.2% BSA, 0.02% soybean-trypsinnhibitor. To determine the extent of cell-cell contact dissoci-tion, we used 2 independent methods. Living acini werencubated for up to 40 minutes with buffer alone or withuffer containing 2 �g/mL human neutrophil elastase or 5g/mL pancreatic elastase (Calbiochem, San Diego, CA) and

hen fixed for morphologic studies. Alternatively, the biovol-
me of living acini was determined. This assay is based on the c
isintegration of cell-cell contacts that permits individualcinar cells to dissociate from the acinus. Accordingly, theiovolume of intact acini (between 3 and 80 cells or a sphereith a diameter of 24–80 �m) decreases when cell contactsissociate, whereas the biovolume proportion of individualingle cells (mean diameter, 17�m; range, 11–23 �m) increas-s.25 Suspensions of freshly prepared acini were diluted (1:200) inltered (particle free) buffer, and the biovolume ratio was deter-ined with a cell analyzing system (CASY I; Schärfe Systems,eutlingen, Germany), which is based on resistance measure-ents with pulse-surface analysis. Measurements during incuba-

ion with the above reagents were made for single cells (11–23m) and intact acini (24–80 �m) and the results expressed asercentage of control incubations with buffer alone. Graphs in-icate the means of 3 or more experiments in each group � SD.25

Immunofluorescence microscopy cells of the human pancre-tic ductal adenocarcinoma cell line PaTu-8988-S were cul-ured on chamber slides in DMEM (10% FCS, 1% L-glu-amine) at 5% CO2 to subconfluency and further incubatedith either purified human neutrophil elastase (5 �g/mL) orurified porcine pancreatic elastase (5 �g/mL) for up to 120inutes. Isolated rat pancreatic acini freshly prepared by col-

agenase digestion were treated with human neutrophil elas-ase for 120 minutes (20 �g/mL) at 37°C. Cells were washedwice in PBS and fixed in 4% PBS-buffered formaldehydeolution for 30 minutes, followed by membrane permeabiliza-ion in 0.1% Triton X-100/PBS buffer for 1 minute. Nonspe-ific antibody binding was blocked in PBS/BSA 1% wt/vol for

hour, followed by overnight incubation with the primaryntibody at a concentration of 5 �g/mL at 4°C. Primaryntibody binding was detected with an isospecies-specific sec-ndary antibody conjugated to fluorescein isothiocyanate don-ey anti-mouse IgG (1:100, lot 39656) or Cy 3-conjugatedoat anti-rabbit IgG (1:200, lot 42925) (Dianova, Hamburg,ermany). Under otherwise identical conditions, controls were

ncubated with either species-specific nonimmune serum, pu-ified IgG, or without primary antibody, and images wereaken under the same exposure, brightness, and contrast set-ings. Fluorescence microscopy was performed on a high-esolution Nikon Improvison confocal imaging system (Ni-on, Waxford, United Kingdom).Sections (2–4 �m) of each paraffin-embedded tissue sample

ere deparaffinized with xylene and rehydrated throughraded alcohol into distilled water. The sections were micro-aved in 10 mmol/L citrate buffer (pH 6) for 5 minutes at 900

and for 10 minutes at 500 W. Slides were then washed inris-buffered saline (5 mmol/L Tris-HCI, 0.3 mol/L NaCI2,H 7.4). All further steps were performed as described above.he primary antibody mouse anti-rat CD45 (5�g/mL) wasrecoupled to fluorescein isothiocyanate. For morphometricvaluation of the extent of pancreatic leukocyte infiltration, ainimum of 10 microscopic fields from each animal (n � 5)ere randomly photographed at a fixed magnification (63),

nd micrographs with positive evidence for CD45-positive
ells (either intravascularly or in the interstitial space) were

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sed for further analysis. Results were expressed as number ofD45-positive cells per standard surface area � SEM.

Electron Microscopy

Small blocks (2 mm in diameter) of rat pancreaticissue from pancreatitis and control animals were fixed in 5%wt/vol) paraformaldehyde/0.2 mol/L piperazine-N,N=-bis2-ethanesulfonic acid], pH 7.0, cryoprotected with polyvi-ylpyrollidine/sucrose, and frozen in liquid nitrogen. Ultra-hin frozen sections (60 nm) were prepared using a LeicaBensheim, Germany) Cryo-ultramicrotome (block tempera-ure, �110°C; knife temperature, �100°C). The sections onormvar-coated copper grids were blocked with PBS, 5% (wt/ol) fetal calf serum (FCS; Life Technologies, Rockville, MD),H 7.4, and then incubated with mouse monoclonal anti-E-adherin antibody (1:10–1:30; Transduction Laboratories,lone 36) for 45 minutes at room temperature. After washingith PBS, the sections were incubated with 10-nm gold-conju-ated goat anti-mouse antibody (dilution 1:10; Dianova, Ham-urg, Germany), washed again with PBS and water, and subse-uently contrasted and embedded by incubation withethylcellulose/uranyl acetate on ice (9:1 mixture of 2% meth-

lcellulose and 4% uranyl acetate). Samples were examined on ahilips 400 electron microscope (Hamburg, Germany). Pancre-tic tissue was also fixed in 2% formaldehyde/2% glutaraldehydeor Epon embedding, and then osmium, uranyl, and lead-con-rasted for EM of thin sections.26

Substrate and Inhibitor Specificity

Measurements of inhibitor specificity were initiatedfter adding 10 mU/mL porcine pancreatic elastase, humaneutrophil elastase, bovine pancreatic trypsin, or bovine pan-reatic chymotrypsin to the PMN elastase inhibitor. Enzymectivity was quantitated by substrate cleavage of 10 �mol/Leutrophil elastase substrate AMC-(MeOSuc-Ala2-Pro-Val),lastase substrate R110-(CBZ-Ala4)2, trypsin substrate R110-CBZ-Ile-Pro-Arg)2, or chymotrypsin substrate AMC-(Suc-la2-Pro-Phe). Measurements were performed in 100 mmol/Lris, pH 8, and 5 mmol/L CaCl2 buffer. The rise in fluores-ence was monitored over 10 minutes at 37°C using a micro-late fluorescence reader (SPECTRAmax GEMINI, Molecularevices, Sunnyvale, CA) at an excitation wavelength of 485m, a cut-off at 515 nm, and an emission of 530 nm. Theavelength combination 340 nm, 420 nm, 460 nm was used

or measurements of the AMC-substrates. The inhibitory effectn 10 mU/mL myeloperoxidase was measured in the presencef 0.53 mmol/L of the substrate o-dianisidine and 0.15mol/L hydrogen peroxide over 10 minutes at 30°C at aavelength of 460 nm and in 50 mmol/L potassium phosphateuffer at pH 6. The effect on 10 U/mL �-amylase was moni-ored using a commercial kit (Roche, Ingelheim, Germany)ver 10 minutes at 37°C at 405 nm and in buffer containingodium chloride (150 mmol/L). Enzyme activities were mea-ured under the above conditions with concentrations of PMNlastase inhibitor from 5 pmol/L to 500 �mol/L diluted in
MSO. Background fluorescence or absorption of uncleaved p
ubstrates was subtracted, and results were shown as percent-ge activity in the presence or absence of the inhibitor.

Myeloperoxidase Activity in Lung andPancreatic Homogenates

Tissue was homogenized on ice in 20 mmol/L potas-ium phosphate buffer (pH 7.4) and centrifuged for 10 min-tes at 20,000g at 4°C. The pellet was resuspended in 50mol/L potassium phosphate buffer (pH 6.0) containing 0.5%

etyltrimethylammoniumbromide. The suspension was frozen/hawed 4 times, sonicated twice for 10 seconds, and centri-uged at 20,000g for 10 minutes at 4°C. MyeloperoxidaseMPO) activity was assayed after mixing 50 �L supernatant in00 �L of 50 mmol/L potassium phosphate buffer (pH 6)ontaining 0.53 mmol/L O-dianisidine and 0.15 mmol/L

2O2. The initial increase in absorbance at 460 nm waseasured at room temperature with a Dynatech MR 5000lisa reader (Eningen, Germany). The results are expressed innits of MPO activity on the basis of 1 unit being able toxidize 1 �mol H2O2 per minute per milligram pancreaticrotein.27 Bars indicate mean values in mU MPO activity perilligram pancreatic protein � SEM from 3 or more animals

er time point.

Elastase Activity in PancreaticHomogenates After PMNElastase Inhibitor Treatment

Tissue was homogenized on ice in 100 mmol/L Tris,H 8, and 5 mmol/L CaCl2 and centrifuged for 10 minutes at0,000g at 4°C. Protein content was determined according tohe method of Bradford.

Ten micromolars R110-(CBZ-Ala4)2 substrate (final concen-ration) and 1 �g protein were incubated in 150 �L finalolume, at an excitation wavelength of 485 nm and an emis-ion wavelength of 530 nm, at 37°C. Initial rates of substrateydrolysis were measured in arbitrary fluorescence units perinute. Enzyme activity was calculated as units/milligramsith purified elastase as an internal standard, and activity was

et in relation to cerulein-treated animals.

Determination of Pancreatic Water Content

The water content of the pancreas was quantified byomparing the weight of the freshly harvested organ (weteight) with the weight of the same tissue after desiccation at60°C for 24 hours (dry weight). The results were expressed asercentage water content.

Amylase Serum Levels

Blood was collected by aortic puncture during death ofhe animals. Following centrifugation, serum was stored at20°C until assayed. Serum �-amylase was measured using

,6-ethylidene-(G7)-1-4-nitrophenyl-(GI)-�, D-maltoheptao-ide as substrate (EPS method) according to the manufacturer’s
rotocol (Roche, Ingelheim, Germany).

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Data Presentation and Statistical Analysis

Data in graphs were expressed as means � SEM (orSD as stated in the text). Statistical comparison of groups at

arious time intervals was done by Student t test for indepen-ent samples as well as Mann-Whitney rank sum test ifndicated using SigmaStat for Windows (SPSS Inc., Chicago,L). Differences were considered significant at a level of P 05. Data presentation was performed with SigmaPlot for

indows (SPSS Inc., Chicago, IL).

Results

Subcellular Localization and Redistributionof E-Cadherin During the Course ofAcute Pancreatitis

Infusion of supramaximal concentrations of cer-lein into male Wistar rats resulted in hyperamylasemia,nterstitial pancreatic edema, and intracellular vacuoliza-ion with a maximum extent 6 hours after the start of thexperiment and an almost complete resorption of thedema after 48 hours. We were interested in the fate of-cadherin in the process of edema formation and per-

ormed electron microscopic analysis of control and cer-lein-infused animals to see whether and how E-cadherinxpression and cellular localization were altered in thearly course of the disease process. Immunogold labelingevealed that E-cadherin expression was strictly mem-rane associated at adherens junctions and interdigita-ions of adjacent cells in control tissue (Figure 1A–C).upramaximal secret-gogue stimulation, however, resulted in a massive ac-umulation of interstitial fluid, a dissociation of cell-cellontacts, and redistribution of E-cadherin from cell con-acts and the lateral cell membrane into the cytoplasmnd interstitial space (Figure 1D and E). Twelve hoursfter the onset of acute pancreatitis, E-cadherin wasound in the cytoplasm and in lysosomes (Figure 1F and, asterisks), which indicates translocation and degrada-

ion. Furthermore, it was found in close proximity withndoplasmic reticulum (ER) elements, which may indi-ate production of newly synthesized E-cadherin (FigureG). Vacuolization, edema formation, and E-cadherinedistribution were not observed in saline-infused controlnimals.

E-Cadherin Cleavage During Pancreatitis

When pancreatic homogenates were assayed for-cadherin expression by Western blot analysis using aonospecific C-terminal antibody, only a single 120-

ilodalton band was detected in control animals (FigureH, 0 hours). As early as 1 hour after the onset of
upramaximal cerulein stimulation, an approximately (
5-kilodalton smaller variant of E-cadherin appeared.fter 4 hours of supramaximal cerulein treatment, the

ntibody detected the newly synthesized precursor E-adherin of approximately 130 kilodaltons, which wasost prominent after 12 hours of supramaximal secre-

agogue stimulation. This was confirmed by semiquan-itative RT-PCR in which an increase of the E-cadherinessenger RNA (mRNA) transcript was found after 4

ours of supramaximal cerulein stimulation (not shown).y subjecting immunoprecipitated E-cadherin to enzy-atic deglycosylation, we ruled out that the smaller-cadherin fragment of 105 kilodaltons represents de-lycosylated E-cadherin (not shown). Immunoprecipita-ion of the 105-kilodalton E-cadherin fragment, usingntibodies specifically recognizing either the C-terminusr the N-terminal peptide sequence, revealed that E-adherin is cleaved at the extracellular region of therotein during early pancreatitis (Figure 1H).

Identification of the E-CadherinCleavage Site

To locate the cleavage site within the E-cadherinortion, we immunoprecipitated the fragment after 4ours of cerulein stimulation from pancreatic homoge-ate and subjected the material to Edman peptide se-uencing. The starting amino acid sequence of the frag-ent was GARIATLK, which matches amino acids 394

o 401 in the EC-3 domain of E-cadherin (protein bankccession number Q9R0T4; Figure 2). A scan of the-cadherin sequence for the occurrence of patterns androfiles stored in the PABASE database (Omiga 2.0,adison, Wisconsin) identified 11 proteases that could

leave E-cadherin in this position. Four of these proteasesere known to be expressed in either pancreatic tissue ory inflammatory cells: (1) A carboxypeptidase, known asathepsin IV, an exopeptidase that hydrolizes Z-Glu-Tyrnd which has not previously been cloned or purifed28;2) the metalloendoproteinase Meprin A, which is mainlyxpressed in the small intestine and known to degradeollagen IV29; (3) lysosomal cysteine peptidase cathepsin30; and (4) polymorphonuclearelastase (leukocyte elas-

ase E:C. 3.4.21.37). The latter belongs to the chymo-rypsin family of serine proteases and is highly expressedn the azurophilic granules of PMN leukocytes (2 pg/ell),31 which have been reported to accumulate in theancreas during acute pancreatitis.32

E-Cadherin as Substrate forLeukocyte Elastase

In accordance with our initial hypothesis, weested whether proteases released by inflammatory cells
now the most abundant and thus probable would be

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igure 1. Ultrastructural localization of E-cadherin. Tissue blocks from rats treated with supramaximal concentrations of cerulein or saline forp to 12 hours were fixed, resin embedded, and labeled with antibodies directed against the intracelluar domain of E-cadherin (5-nm gold).epresentative electron micrographs from control animals are shown in A–C, whereas D and E represent micrographs from animals infused withupramaximal concentrations of cerulein for 4 hours. In F and G, duration of cerulein infusion was 12 hours. Bars indicate 1 �m. In control tissue,-cadherin is exclusively located at the cell membrane and at cell-cell contacts and interdigitations of epithelial cells (A–C). After 4 hours ofupramaximal cerulein stimulation, E-cadherin label has separated from junctions and redistributed to the cytoplasm (bold arrows in D and E)r the widened interstitial space between acinar cells whose cell adhesions have dissociated (fine arrows). After 12 hours, E-cadherin label isetected in the cytoplasm, ER (arrows in G), and lysosomes (asterisks, F and G). Incubation of control sections with nonspecific IgG resulted ino background gold labeling (data not shown). (H) Western blots of E-cadherin in pancreatic homogenates between 0 and 48 hours ofxperimental pancreatitis using antibody directed against the E-cadherin C-terminus and, for immunoprecipitation (2 lower panels in H), againsthe C-terminus and the N-terminus. Note E-cadherin cleavage (105-kilodalton band) from the N-terminus as early as 1 hour after the start of the
erulein infusion and the E-cadherin precursor (130-kilodalton band) most prominent after 12 hours.

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MN elastase) or generated by pancreatic exocrine cellseg, activated digestive zymogens) cause E-cadherin pro-essing at the identified cleavage site. We incubated cellsf the epithelial pancreatic cancer cell line PaTu-8889-S,hich do not express digestive protease zymogens, withMN elastase. In cells treated with purified PMN elas-ase (2 �g/mL) for up to 120 minutes, an identicalhortened E-cadherin fragment as seen during the coursef cerulein pancreatitis was detected (Figure 3A). Toxclude degradation of E-cadherin by endogenously ex-ressed proteases, even in the presence of abundant pro-ease inhibitors in the lysis buffer, cells were lysed in theresence (0�) and absence (0�) of PMN elastase. Tonsure the specificity of the cleavage product found in theysates, we performed immunoprecipitation experimentssing homogenates from untreated control cells incu-ated with PMN elastase after immunoprecipitation asell as from cells treated with PMN elastase (2 �g/mL)efore lysis (Figure 3B). Under both conditions, themaller 105-kilodalton variant of E-cadherin was detect-ble, and, by Edman peptide sequencing, we confirmedhat the fragment generated under PMN elastase treat-ent was identical to the one detected in acute pancre-

titis.The substrate specificity of PMN elastase is very sim-

lar to that of pancreatic elastase, which is known to bectivated intracellularly during pancreatitis and may

igure 2. Schematic representation of E-cadherin domain structurend localization of the PMN-elastase cleavage site. The extracellularepeat units of E-cadherin EC1 through EC5 are preceded by a short,-terminal signal sequence that is cleaved during maturation of E-adherin. The HAV region (light grey box) of EC-1 is required foromotypic interactions. TM indicates the transmembrane region,hereas the proximal intracellular region (dark grey box) binds120ctn, and the distal region (intermediate grey box) binds either-catenin or �-catenin. Peptide sequencing of the cleaved E-cadherinetected after supramaximal cerulein stimulation identified aminocid Gly-394 at the N-terminus of the EC-3 domain. Proteolytic cleav-ge therefore must occur between Val-393 and Gly-394, suggestingMN-elastase (leukocyte-elastase) as the most likely protease re-ponsible for extracellular shedding.

ubsequently be released into the interstitial space. Nev- u

rtheless, when we incubated PaTu-8889-S cells withurified pancreatic elastase (up to 5 �g/mL), we couldot detect an E-cadherin cleavage product (Figure 3Cnd D). After treatment of PaTu-8889-S cells with pan-reatic elastase (5 �g/mL), E-cadherin displayed a strongembrane localization on fluorescence microscopy (Fig-

re 3E), resembling the distribution in untreated cellsnot shown). Incubation with PMN elastase (2 �g/mL),owever, induced a loss of membrane-bound E-cadherinnd its redistribution to the cytoplasm (Figure 3F).

Ex Vivo Cleavage of E-Cadherin on AcinarCells by PMN Elastase

Supramaximal stimulation of freshly isolated pan-reatic acini, functional secretory units of 5 to 80 exo-rine cells, with either cerulein or CCK (10�6 mol/L)eads to intracellular protease activation, cell injury, andelease of activated proteases into the supernatant. Thisx vivo model mimics several of the earliest changes inhe onset of acute experimental pancreatitis. Therefore,o distinguish further between activities of exogenousPMN elastase) and endogenous (activated digestive zy-ogens) proteases on the cadherin/catenin complex, pan-

reatic acini were freshly isolated and incubated withither CCK 10�6 mol/L, PMN elastase, or a combinationf both. In addition, we incubated the cells with pan-reatic elastase at a concentration of 5 �g/mL. Subse-uently, immunoprecipitation studies were performed todentify the generation of the E-cadherin fragments.nterestingly, incubation with supramaximal concentra-ions of CCK alone, although followed by massive intra-ellular protease activation (not shown), did not cause-cadherin cleavage (Figure 4A and B). Incubation withancreatic elastase did also not result in E-cadherin cleav-ge. Incubation with PMN elastase alone, on the otherand, caused significant formation of the 105-kilodalton-cadherin cleavage product accounting for up to 55% ofhe total E-cadherin content. Coincubation with CCKnd PMN elastase did not further add to the cleavage of-cadherin (Figure 4A and B). These results clearly dem-nstrate that endogenous pancreatic proteases in gener-l—and pancreatic elastase in particular—are neitherequired nor involved in the cleavage of E-cadherin inancreatic acinar cells upon supramaximal secretagoguetimulation.

The Role of PMN Elastase in theDissociation of Cell-Cell Contacts and theInternalization of E-Cadherin

The extracellular EC-1 domain contains an HAVeptide sequence and has been suggested to be a prereq-
isite for the maintenance of intact cell-cell contacts at

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dherens junctions. In isolated pancreatic acini, whichepresent fully functional secretory units with intacttimulus-secretion coupling, cell-cell contacts remain in-act for up to 4 hours in vitro. Over this period, theirostisolation diameter and volume remain fairly stable.o study the effect of PMN elastase on acinar cell-cellontact integrity, we incubated freshly isolated rat pan-reatic acini for up to 40 minutes with leukocyte elastase

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™igure 3. N-terminal cleavage of E-cadherin by PMN-elastase in vivo. Ininutes and subsequent Western blot analysis of total lysates (A, lefirected against the C-terminus revealed an E-cadherin fragment idtimulation in vivo (A, right panel). Figure 3B, right panel shows thelastase for up to 120 minutes. 0� symbolizes lysis in the presenceith pancreatic elastase in a time- and concentration-dependent maipitation studies and subsequent Western blotting for E-cadherin. Immancreatic elastase (5 �g/mL) for 120 minutes showed normal E-cadh

ncubation with leukocyte elastase (5 �g/mL for 120 minutes) resu

igure 4. E-cadherin cleavage on pancreatic acini. Pancreatic aciniere freshly isolated by collagenase digestion and incubated either in

he presence of CCK 10�6 mol/L or with pancreatic elastase (5g/mL), with leukocyte elastase (5�g/mL), or with a combination of

eukocyte elastase and cerulein for 120 minutes at 37°C. Salinencubation served as control. Subsequently, pancreatic acini wereubjected to immunoprecipitation and analyzed for the presence ofhe E-cadherin fragment. Incubation of pancreatic acini with CCKlone or pancreatic elastase did not cause E-cadherin cleavage (A and), whereas incubation with leukocyte elastase cleaved �55% of-cadherin. Incubation of pancreatic acini with a combination of leu-ocyte elastase and CCK did not increase E-cadherin cleavage overhe extent obtained with leukocyte elastase alone. Densitometricnalysis of the E-cadherin cleavage product was undertaken usingestern blots from 3 different experiments, and error bars representeans in percentage � SEM.

-cadherin.

r pancreatic elastase. To determine the extent of cellontact dissociation, we measured the release of singleells (diameter, 11–23 �m) from intact acini vs themount of remaining intact acini (diameter, 24–80m). An increase of single cells in parallel with a declinef the larger intact acini reflected a dissociation of cellontacts, which could be confirmed by confocal micros-opy. During incubation with buffer alone serving asontrol, or with pancreatic elastase, acini remained stablehroughout the entire experiment, and neither the pro-ortion of single cells (Figure 5A) nor the number ofntact acini (Figure 5B) changed. In immunofluorescencetudies of these acini, E-cadherin displayed a clearlyembrane-associated localization and nearly absent cy-

oplasmic expression (Figure 5C). Incubation with pan-reatic elastase did not change the subcellular localiza-ion of E-cadherin (data not shown). Incubation withMN elastase induced a rapid dissociation of adherens

unctions between pancreatic acinar cells, resulting in theeneration of single cells (Figure 5A) and a progressiveecline of intact acini. The prominent cytoplasmic label-ng of E-cadherin in these cells after PMN elastase in-ubation confirms its redistribution to the cytoplasmFigure 5D). When we investigated homogenates of pan-reatic acini for ubiquitinilation of E-cadherin and itsorting into the proteasome pathway, we found a rapidurnover of E-cadherin after PMN elastase incubationnot shown). These data give evidence that incubation ofcinar cells with PMN elastase is entirely sufficient toissociate cell-cell contacts and redistribute E-cadherinrom the membrane to the cytoplasm.

Early Leukocyte Transmigration Into thePancreas During Pancreatitis

To study whether leukocyte infiltration parallels-cadherin cleavage, we infused animals with supramaxi-al concentrations of cerulein and measured PMN elas-

ase protein expression (Figure 6A) as well as MPOctivity (Figure 6B) as markers of leukocyte infiltrationn pancreatic tissue homogenates at distinct intervalsfter pancreatitis onset. As early as 1 hour after the onset

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™tion of PaTu-8889-S cells with PMN-elastase (2 �g/mL) for up to 120el) and immunoprecipitates (B, left panel) using E-cadherin antibodyl to the one observed after 60 minutes of supramaximal cerulein

lt of an incubation of immunoprecipitated E-cadherin with leukocyteukocyte elastase (2 �g/mL). PaTu-8889-S cells were also incubated(C and D), but no E-cadherin fragment was detected in immunopre-uorescence analysis for E-cadherin of PaTu-8889-S cells treated with

abeling at intact cell contacts and the plasma membrane (E), whereasn cell-cell contact dissociation and progressive internalization (F) of

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f acute pancreatitis, a 10-fold increase of MPO activityompared with saline-infused control animals was foundFigure 6B). Maximal MPO activity was measured 12ours after the onset of cerulein pancreatitis with a

igure 5. Cell contact dissociation after incubation with leukocyte ela) vs intact acini (diameter, 24–80 �m; B) after incubation with leukinutes. Incubation with leukocyte elastase induced a rapid dissoc

ncrease in the proportion of single cells (A, filled lozenges) and a paraf acini during incubation with buffer alone or with pancreatic elastasingle cells (A) nor of intact acini (B) changed. We also studied paraformontrol acini and acini incubated with pancreatic elastase (not shown)ontacs (C). After leukocyte elastase incubation (D), cell contacts wembrane to the cytosol.

ubsequent decline until 48 hours of supramaximal cer- p

lein stimulation—a time point when cell-cell contactsave reassembled. In parallel to MPO activity, also PMNlastase, as determined by immunoprecipitation and

estern blotting, peaked at 12 hours after the onset of

. We quantitated the biovolume of single cells (diameter, 11–23 �m;elastase (2 �g/mL) or pancreatic elastase (5 �g/mL) for up to 40of cell-cell contacts within the acini as indicated by a progressive

ecline in the number of intact acini (B, filled lozenges). The biovolumeained stable throughout the experiment, and neither the numbers ofhyde-fixed acini for E-cadherin localization and change in morphology.layed a clear membrane-bound E-cadherin staining and intact cell-cellund largely dissociated, and E-cadherin was redistributed from the

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he first 60 minutes (Figure 6A). EM micrographs re-ealed a significant transmigration of inflammatory cellsrom small blood vessels into the interstitial space at thatime (Figure 6C and D). Furthermore, when we quanti-ated leukocyte infiltration into pancreatic tissue duringhe course of acute pancreatitis, we found a significantncrease in CD45-positive leukocytes, outside of bloodessels, that had transmigrated into the intercellular

igure 6. Leukocyte infiltration of pancreatic tissue during experimen-al pancreatitis. Protein amounts of PMN leukocyte elastase as de-ermined by immunoprecipitation and Western blotting (A) as well asyeloperoxidase enzymatic activity (B) were already increased inancreatic tissue after the first hour of experimental pancreatitis. Theaximum increase of both enzymes was seen at 12 hours. Datahown are representative of 3 or more experiments (B, indicateseans � SEM). In parallel to the increased amounts of leukocytearker enzymes in tissue homogenates, leukocyte migration fromlood vessels into the interstitial space (circles in C) or in betweenancreatic acinar cells (circles in D) is detectable in EM.

pace (Figure 7A and B). As early as 1 hour after the t

nset of the disease, CD45 labeling revealed a 6-foldncreased number of leukocytes in the space betweenancreatic acini as illustrated in the right panel of FigureB. These results indicate that PMN elastase is capablef cleaving E-cadherin at the identified site in vivo and initro and that neutrophils, which secrete PMN elastase,ppear at the site at which E-cadherin cleavage occurs.

Specificity of PMN Elastase Inhibition

To determine whether PMN elastase inhibitionould prevent cell contact dissociation as well as E-

igure 7. Quantitative leukocyte infiltration during experimental pan-reatitis. Immunofluorescence labeling of common leukocyte antigenCD45) was done in paraffin sections of pancreatic tissue over thentire time course of pancreatitis. Already, after 1 hour, a significantncrease of CD45-positive leukocytes in the pancreatic tissue can behown (A). The left micrograph (B) shows the labeling of untreatedissue, whereas the right panel is representative for 1 hour of ceruleinreatment. CD45-positive fluorescent cells are marked with circles.he insert in the right panel shows at higher magnification that theytoplasm, rather then the nuclei, of inflammatory cells are CD45ositive. For morphometric evaluation of the extent of leukocyte infil-ration in the pancreas, 10 micrographs from each animal (n � 5)ere randomly photographed at a fixed magnification (63), and

issue-infiltrating CD45-positive cells were quantitated.

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adherin cleavage and thus have a beneficial effect on theourse of pancreatitis, we used a PMN elastase inhibitorn in vivo experiments. To determine the specificity ofhe inhibitor for PMN elastase, rather than for otherancreatic digestive enzymes, which are known to bectivated or released during pancreatitis, we performed aeries of in vitro experiments. We therefore compared thenhibitory capacity of a specific neutrophil elastase pep-ide inhibitor coupled to a chloro-methyl-coumarin res-due (MeOSuc-Ala-Ala-Pro-Ala-CMK) on trypsin, chy-otrypsin, pancreatic elastase, human neutrophil

lastase, myeloperoxidase, and amylase. The inhibitor atoncentrations ranging from 5 pmol/L to 500 �mol/Lid not affect the activity of either trypsin, chymotryp-in, myeloperoxidase, or amylase. The inhibitory capacityor human neutrophil elastase was nearly half log higherhan for pancreatic elastase. The IC 50 for PMN elastaseas calculated to be 2.8 � 1.7 �mol/L, whereas the IC0 for pancreatic elastase was calculated to be 15.2mol/L � 2.5 �mol/L (Figure 8).

PMN Elastase Inhibition PreventsE-Cadherin Cleavage and ReducesLeukocyte Infiltration During Pancreatitis

To investigate the role of PMN elastase-mediated

igure 8. PMN-elastase inhibitor specificity in vitro. Myeloperoxidase10 mU/mL), human neutrophil elastase (10 mU/mL), pancreaticlastase (10 mU/mL), trypsin (10 mU/mL), chymotrypsin (10 mU/L), and amylase (10 U/mL) were incubated with various concentra-

ions of inhibitor ranging from 5 nmol/L to 500 �mol/L, and enzymectivities were detected using specific fluorogenic or chromogenicubstrates in a multiplate fluorescence or absorbance reader. Activityrom 3 or more different experiments is expressed as the percentagef enzyme activity detected in the absence of the inhibitor. Humaneutrophil elastase (PMN-elastase)—and to a somewhat lesser ex-ent pancreatic elastase—was inhibited by the PMN-elastase inhibitorith 50% inhibition at 2.8 �mol/L (PMN-elastase). Trypsin, myeloper-xidase, chymotrypsin, and amylase were not significantly affected by

ncubation with the inhibitor.

-cadherin cleavage and cell-cell contact dissociation, we m

nhibited PMN elastase in vivo in rats during experi-ental pancreatitis. To this end, we treated animals with

he above neutrophil elastase peptide inhibitor coupledo a methyl-coumarin residue (MeOSuc-Ala-Ala-Pro-la-CMK), together with supramaximal concentrationsf cerulein for 2 hours. E-cadherin cleavage productsere identified from pancreatic tissue homogenates by

mmunoprecipitation and Western blotting. Althoughupramaximal concentrations of cerulein alone induced-cadherin shedding, the intravenous administration ofhe inhibitor reduced E-cadherin cleavage almost com-letely (Figure 9A). MPO activity in pancreatic tissue

igure 9. Inhibition of PMN-elastase activity prevents leukocyte trans-igration during pancreatitis. Infusion of male Wistar rats with cer-lein (10 �g/kg per hour) in the presence of the leukocyte elastasenhibitor (50 �mol/L) still resulted in the induction of pancreatitis butrevented cleavage of E-cadherin (A) and significantly reduced theranslocation of leukocytes into pancreatic tissue as indicated byeduced MPO activity in pancreatic homogenates (B). C The equivalentnhibitor concentration reduced PMN elastase activity by 80% in pan-reatic homogenates. Data shown are representative of 5 experi-
ents (B and C represent means � SEM).

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rom animals treated with the PMN elastase inhibitoras found to be reduced, and thus the number of infil-

rating inflammatory cells during pancreatitis (the meanPO in cerulein treated animals was 6.1 � 1.5 [SD]U/mg protein vs 3.3 � 1.6 [SD] mU/mg protein in

nimals treated with cerulein plus PMN inhibitor; 95%onfidence interval for the difference of the means: 0.9 to.7 mU/mg protein, P � .009; n � 6; Figure 9B).dditionally, and as an internal control, we determined

he inhibitor capacity for PMN elastase in vivo. PMNlastase-induced substrate cleavage of R110-(CBZ-Ala4)2

n pancreatic homogenates after cerulein application waseduced to 20% (Figure 9C). This clearly indicated thatMN elastase-mediated E-cadherin cleavage is a prereq-isite for the dissociation of adherens junctions andeukocyte infiltration into the pancreatic tissue duringhe early course of pancreatitis. Inhibition of PMN elas-

igure 10. Inhibition of leukocyte elastase results in amelioration of aour) in the presence of the leukocyte elastase inhibitor (50 �mol/ystemic inflammatory response as indicated by reduced MPO activityn D illustrates leukocyte extravasation into pancreatic tissue after cef PMN elastase inhibitor and cerulein. Size bars indicate 200 �m. DEM).

ase activity, on the other hand, can prevent inflamma- A

ory cells from entering the pancreas during an episode ofcute pancreatitis.

Leukocyte Elastase Inhibition AmelioratesLocal and Systemic Injury in Pancreatitis

To investigate whether inhibition of PMN elas-ase could ameliorate the course of acute pancreatitis, weetermined serum amylase, pancreatic edema, and my-loperoxidase activity in the lungs after in vivo admin-stration of the inhibitor (Figure 10). Serum amylaseevels were significantly reduced after inhibitor treat-ent (amylase activity in cerulein-treated animals

6,962 mU � 1257 [SD] mU vs 9177 mU � 1638 [SD]U in cerulein plus PMN elastase inhibitor-treated an-

mals, n � 5; 95% confidence interval for the differencef the means: 4474–11,096, P � .003) (Figure 10A).

pancreatitis. Infusion of male Wistar rats with cerulein (10 �g/kg per2 hours led to a significant decrease in serum amylase (A), in thelungs (B), and in pancreatic edema (C). Immunolocalization of CD45administration for 2 hours (insert) and the combined administrationhown are representative of 5 experiments (A–C represent means �

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reated animals, 85.7% � 0.9% [water content in per-entage �SD] vs 79.8% � 0.5% in animals treated witherulein plus PMN elastase inhibitor; n � 5; 95% con-dence interval for the difference of the means: 4.9 to 7; .001; Figure 10C). The reduced myeloperoxidase

ctivity in the lungs suggested that, besides local pan-reatic damage, the systemic inflammatory response waseneficially affected (MPO in lung tissue in cerulein-reated animals was 13.4 � 3.2 [SD] mU/mg protein vs.6 � 1.9 mU/mg protein in animals treated witherulein plus PMN elastase inhibitor; P .001; 95%onfidence intervals for the difference of the means: 3 to.7; n � 8; Figure 10B). To exclude a mere accumulationf leukocytes in pancreatic vessels to account for thencrease in MPO activity in pancreatic tissue homoge-ates, we performed immunolabeling of CD45 (commoneukocyte antigen) in paraffin sections. Already, after 2ours, CD45 labelling revealed an increased leukocyteransmigration into pancreatic tissue as illustrated byigure 10D. In summary, our data lead to the conclusionhat administration of a PMN elastase inhibitor improveshe clinical outcome of acute cerulein-induced pancreati-is.

Discussion

The regulatory mechanisms involved in cadherin-ependent cell adhesions are complex and not completelynderstood. Although it is well established that proteinsf the cadherin/catenin complex play an important rolen embryonic development, in tissue morphogenesis, andn malignant tumor invasion, information regarding theechanisms that control the function of the cadherin/

atenin complex in inflammation is sparse. We havereviously shown that in vivo administration of secreta-ogues (cholecystokinin or acetylcholine) at concentra-ions that are in excess of those required for a maximalecretory response induces cellular changes in the rat orouse pancreas that involve the dissociation of adherens

unctions,9 a complex cascade of tyrosine phosphoryla-ion events regarding cell adhesion proteins,25 a disas-embly of the cytoskeleton,33 and a disturbance of theesicular transport within the acinar cell.22 Because thesehanges are completely reversible within 48 hours inivo, we used the animal model here to study the mech-nisms involved in cell-cell contact dissociation and re-ssembly. When we studied E-cadherin expression in theancreas over the first 48 hours of experimental pancre-titis, we detected an additional, unpredicted 105-kilo-alton E-cadherin band, which appeared as early as 1our after disease induction. Because it is known that
-cadherin is highly N-glycosylated in the extracellular w
omain34 and has an O-glycosylation site in the intra-ellular domain,35 we tested deglycosylation as a possibleause for the smaller E-cadherin fragment but found nondication for any deglycosylation process. Immunopre-ipitation experiments and peptide sequencing revealedhat the newly formed molecule is a cleavage product of-cadherin that lacks the extracellular EC-1 and EC-2omains. Although classical cadherins consist of 5 re-eated domains (EC-1 to EC-5) with great internal ho-ology in their extracellular segment,36,37 the only site

nvolved in cell contact formation is in the cadherin EC-1omain. This domain includes an HAV amino acid motifhat directly mediates adhesive interactions. We there-ore had to assume that the 105-kilodalton E-cadherinragment identified during pancreatitis is nonadhesive.ur studies on pancreatic acini in which an increase in

he shortened 105-kilodalton E-cadherin was associatedith cell contact dissociation supported this notion.apping of the cleavage site to amino acid 394 and

nalysis of the cleavage motif ruled out caspases or met-lloendoproteinases as possible candidates for the shed-ing of E-cadherin but identified PMN leukocyte elas-ase as most likely responsible for the observed cleavage.n previous cell culture experiments, proteolytic eventsad also been found to be involved in leukocyte trans-igration through an endothelial layer,38,39 although

ittle was revealed about the underlying mechanisms andhe resulting epithelial injury in vivo.13 Proteolyticleavage of E-cadherin as the mechanism by which the05-kilodalton form is generated raised the question ofhether the required proteolytic activity originated from

nvading inflammatory cells (eg, PMN leukocyte elas-ase) or from pancreatic acinar cells, which synthesizeigestive protease zymogens in abundance and are knowno activate them during pancreatitis.15,18–20,27 To distin-uish these, we followed several approaches: (1) In pan-reatic epithelial tumor cells with intact adherens junc-ions but devoid of endogenous expression of anyctivatable digestive proteases, PMN elastase incubationroduced the same 105-kilodalton E-cadherin cleavageroduct as seen in pancreatitis. Nevertheless, the efficacyf PMN elastase cleavage was not 100%, which could bexplained by either a change in biochemical conforma-ion because of detergents used for immunoprecipitationr may indicate that PMN elastase acts in concert withther proteases that are coinhibited in vivo by the PMNlastase inhibitor used here. (2) Incubation of pancreaticcini with PMN elastase resulted in E-cadherin cleavage,n redistribution of the intracellular part of the protein tohe cytoplasm, and in a rapid dissociation of cell-cellontacts. On the other hand, supramaximal stimulation
ith a secretagogue alone, which is known to induce

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mmediate and abundant intracellular protease activa-ion, did not induce E-cadherin cleavage nor did theombination of PMN elastase incubation with secreta-ogue stimulation increase E-cadherin cleavage observedith PMN elastase alone. (3) Pancreatic elastase, a di-estive protease that is activated early in pancreatitis andan be released from acinar cells, had no effect on eitherhe integrity of E-cadherin or on that of cell-cell con-acts. Taken together, these data provide compellingvidence that PMN leukocyte elastase is, indeed, thenzyme that can cleave E-cadherin on adjacent pancreaticcinar cells to dissociate cell-cell contacts and permitnflammatory cells to enter the pancreatic tissue.

Tissue infiltration by inflammatory cells has tradition-lly been regarded as a late event and not as an earlyvent in the course of pancreatitis.21,26 In our study, weave compared the time course of E-cadherin cleavageith that of inflammatory cell infiltration and the pres-

nce of leukocyte products in the pancreas (CD45-posi-ive leukocytes in the interstitial space, pancreatic tissuePO activity, and pancreatic tissue PMN elastase ex-

ression). We were rather surprised to find inflammatoryell infiltration to occur as early as 1 hour after the startf pancreatitis and could confirm this observation by all

lines of evidence. Our data are, however, entirelyonsistent with previously published studies that usedhe same animal model and determined MPO activitiesrom the third hour onward after the start of the ceruleinnfusion.40 To our knowledge, no published reports existn which pancreatic myeloperoxidase levels were deter-ined in the rat at earlier time points during the course

f experimental pancreatitis.One line of evidence suggests that interleukin 8, tu-or necrosis factor �, and MCP-1, which are released

rom macrophages, endothelial cells, and epithelial cells,re the mediators that attract neutrophils to the site ofancreatic injury.41 In this context, it has been shown bythers that a significant increase in the tumor necrosisactor � release from pancreatic acini can be detected asarly as 30 minutes after the start of a supramaximalerulein stimulation and can subsequently attract neu-rophils to the site of inflammation.42 In another line ofvidence, the incubation of isolated neutrophils withurified pancreatic enzymes led to the degranulation ofhe neutrophils and to their transmigration in a matrigelhamber.43 Because intracellular protease activation is aery early event in acute pancreatitis, it is thus feasiblehat activated digestive proteases directly induce theegranulation of neutrophils with a subsequent rise inxtracellular PMN elastase. This scenario would be anlternative pathway to the classical, chemoattraction-
ediated neutrophil infiltration. c
Moreover, it has been demonstrated in different in-ammatory models that E-selectin and P-selectin, thexpression of both of which is a prerequisite for leukocyteransmigration, are up-regulated during the first 30 min-tes of inflammation.44,45 Real-time imaging studies ofeutrophil transmigration through an endothelial layerave further shown that this process takes less than 10inutes.46

Approximately 3 decades ago, Geokas et al47 reportedhat pancreatic tissue obtained from patients who died ofcute hemorrhagic pancreatitis showed substantially in-reased destruction of elastic tissue of intrapancreaticessels and epithelial cells. This would suggest an im-ortant role for leukocyte elastase in the distinctiveascular injury and necrotic parenchyma of acute hem-rrhagic pancreatitis in humans. Increased leukocytelastase activity was also found in cerulein-induced pan-reatitis.48 Circulating levels of neutrophil elastase werehown to correlate with the severity of pancreatitis andere suggested as a reliable parameter to predict the

everity of the disease.49,50 From these data, one couldonclude that inhibition of leukocyte elastase by specificnhibitors would ameliorate the severity of the disease,nd, subsequently, several inhibitors were designed withifferent pharmacologic properties.51,52 Song et al inves-igated the elastase inhibitor, guamerin-derived syn-hetic peptide (GDSP), which improved several param-ters of cerulein-induced acute pancreatitis in the rat.hey also reported effects of GDSP on superoxide for-ation by activated human neutrophils. This report thus

ndicated that neutrophil elastase inhibitors may amelio-ate pancreatitis via effects on the endothelium, whichrevent leukocyte migration from the vasculature intohe interstitial space of the parenchyma.53 Furthermore,reatment of experimental pancreatitis with antineutro-hil serum did not only abolish signs of inflammatoryesponse but also diminished protease activation andmylase serum levels.54 All of this would be in accor-ance with our data concerning the question of whethernhibition of leukocyte elastase would be associated witheduced tissue damage and reduced leukocyte transmi-ration through epithelial cell-cell junctions and there-ore of therapeutic benefit.

Our data must not only be regarded as direct evidenceor a role of inflammatory cells in the initial phase ofancreatitis but also for a PMN elastase-mediated mech-nism by which leukocytes dissociate cell contacts be-ween epithelial acinar cells and transmigrate into theancreatic tissue. In addition, our results point to aromising treatment strategy: PMN elastase inhibitions a therapy for pancreatitis. In patients with a severe
ourse of pancreatitis, as well as those suffering from

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nherited varieties of recurrent pancreatitis, a great ex-ent of tissue damage is attributed to the action ofnflammatory cells that have transmigrated into the pan-reas.55–61 Treatment with a PMN elastase inhibitor,hich, according to our data, can greatly reduce pancre-

tic inflammation irrespective of the initiating triggeringechanism, seems like a promising therapeutic option

or either limiting the extent of tissue injury or forreventing a disease recurrence.We identified PMN elastase-dependent proteolytic

leavage of E-cadherin and subsequent dissociation ofell-cell contacts as a mechanism through which inflam-atory cells transmigrate into the pancreas during an

nflammatory process such as pancreatitis. Targeting thisechanism therapeutically seems like a feasible and

romising treatment option for inflammatory disordersssociated with extensive tissue damage.

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ing sites in cadherin cell adhesion molecules. Cell 1990;61:147–155.

3. Koch AW, Bozic D, Pertz O, Engel J. Homophilic adhesion bycadherins. Curr Opin Struct Biol 1999;9:275–281.

4. Blaschuk OW, Sullivan R, David S, Pouliot Y. Identification of acadherin cell adhesion recognition sequence. Dev Biol 1990;139:227–229.

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9. Lerch MM, Lutz MP, Weidenbach H, Muller-Pillasch F, Gress TM,Leser J, Adler G. Dissociation and reassembly of adherens junc-tions during experimental acute pancreatitis. Gastroenterology1997;113:1355–1366.

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4. Grady T, Mah’Moud M, Otani T, Rhee S, Lerch MM, Gorelick FS.Zymogen proteolysis within the pancreatic acinar cell is associ-ated with cellular injury. Am J Physiol 1998;275:G1010–G1017.

5. Halangk W, Kruger B, Ruthenburger M, Sturzebecher J, AlbrechtE, Lippert H, Lerch MM. Trypsin activity is not involved in prema-ture, intrapancreatic trypsinogen activation. Am J Physiol Gastro-intest Liver Physiol 2002;282:G367–G374.

6. Hofbauer B, Saluja AK, Lerch MM, Bhagat L, Bhatia M, Lee HS,Frossard JL, Adler G, Steer ML. Intra-acinar cell activation oftrypsinogen during cerulein-induced pancreatitis in rats. Am JPhysiol 1998;275:G352–G362.

7. Poch B, Gansauge F, Rau B, Wittel U, Gansauge S, Nussler AK,Schoenberg M, Beger HG. The role of polymorphonuclear leuko-cytes and oxygen-derived free radicals in experimental acutepancreatitis: mediators of local destruction and activators ofinflammation. FEBS Lett 1999;461:268–272.

8. Kruger B, Albrecht E, Lerch MM. The role of intracellular calciumsignaling in premature protease activation and the onset ofpancreatitis. Am J Pathol 2000;157:43–50.

9. Kruger B, Lerch MM, Tessenow W. Direct detection of prematureprotease activation in living pancreatic acinar cells. Lab Invest1998;78:763–764.

0. Kruger B, Weber IA, Albrecht E, Mooren FC, Lerch MM. Effect ofhyperthermia on premature intracellular trypsinogen activation inthe exocrine pancreas. Biochem Biophys Res Commun 2001;282:159–165.

1. Lerch MM, Saluja AK, Runzi M, Dawra R, Steer ML. Luminalendocytosis and intracellular targeting by acinar cells during earlybiliary pancreatitis in the opossum. J Clin Invest 1995;95:2222–2231.

2. Fuchs M, Muller T, Lerch MM, Ullrich A. Association of humanprotein-tyrosine phosphatase with members of the armadillofamily. J Biol Chem 1996;271:16712–16719.

3. Schnekenburger J, Mayerle J, Simon P, Domschke W, Lerch MM.Protein tyrosine dephosphorylation and the maintenance of celladhesions in the pancreas. Ann N Y Acad Sci 1999;880:157–165.

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5. Schnekenburger J, Mayerle J, Krüger B, Buchwalow I, Weiss FU,Albrecht E, Samoilova VE, Domschke W, Lerch WM. PTP and PTPSHP-1 are involved in the regulation of cell-cell contacts at adhe-rens junctions in the exocrine pancreas. Gut 2005. in press.

6. Lerch MM, Saluja AK, Dawra R, Ramarao P, Saluja M, Steer ML.Acute necrotizing pancreatitis in the opossum: earliest morpho-logical changes involve acinar cells. Gastroenterology 1992;103:205–213.

7. Halangk W, Lerch MM, Brandt-Nedelev B, Roth W, RuthenbuergerM, Reinheckel T, Domschke W, Lippert H, Peters C, Deussing J.Role of cathepsin B in intracellular trypsinogen activation and theonset of acute pancreatitis. J Clin Invest 2000;106:773–781.

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3. Jungermann J, Lerch MM, Weidenbach H, Lutz MP, Kruger B,Adler G. Disassembly of rat pancreatic acinar cell cytoskeletonduring supramaximal secretagogue stimulation. Am J Physiol1995;268:G328–G338.

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9. Allport JR, Muller WA, Luscinskas FW. Monocytes induce revers-ible focal changes in vascular endothelial cadherin complex dur-ing transendothelial migration under flow. J Cell Biol 2000;148:203–216.

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4. Gukovskaya AS, Vaquero E, Zaninovic V, Gorelick FS, Lusis AJ,Brennan ML, Holland S, Pandol SJ. Neutrophils and NADPH oxidasemediate intrapancreatic trypsin activation in murine experimentalacute pancreatitis. Gastroenterology 2002;122:974–984.

5. Howes N, Lerch MM, Greenhalf W, Stocken DD, Ellis I, Simon P,Truninger K, Ammann R, Cavallini G, Charnley RM, Uomo G,Delhaye M, Spicak J, Drumm B, Jansen J, Mountford R, WhitcombDC, Neoptolemos JP. Clinical and genetic characteristics of he-reditary pancreatitis in Europe. Clin Gastroenterol Hepatol 2004;2:252–261.

6. Keim V, Bauer N, Teich N, Simon P, Lerch MM, Mossner J.Clinical characterization of patients with hereditary pancreatitisand mutations in the cationic trypsinogen gene. Am J Med 2001;111:622–626.

7. Ellis I, Lerch MM, Whitcomb DC. Genetic testing for hereditarypancreatitis: guidelines for indications, counselling, consent andprivacy issues. Pancreatology 2001;1:405–415.

8. Lerch MM, Ellis I, Whitcomb DC, Keim V, Simon P, Howes N,Rutherford S, Domschke W, Imrie C, Neoptolemos JP. Maternalinheritance pattern of hereditary pancreatitis in patients withpancreatic carcinoma. J Natl Cancer Inst 1999;91:723–724.

9. Lowenfels AB, Maisonneuve P, DiMagno EP, Elitsur Y, Gates LKJr, Perrault J, Whitcomb DC. Hereditary pancreatitis and the riskof pancreatic cancer. International Hereditary Pancreatitis StudyGroup. J Natl Cancer Inst 1997;89:442–446.

0. Lowenfels AB, Maisonneuve P, Whitcomb DC, Lerch MM, Di-Magno EP. Cigarette smoking as a risk factor for pancreaticcancer in patients with hereditary pancreatitis. JAMA 2001;286:169–170.

1. Simon P, Weiss FU, Sahin-Toth M, Parry M, Nayler O, Lenfers B,Schnekenburger J, Mayerle J, Domschke W, Lerch MM. Heredi-tary pancreatitis caused by a novel PRSS1 mutation (Arg-122-�Cys) that alters autoactivation and autodegradation of cationictrypsinogen. J Biol Chem 2002;277:5404–5410.

Received December 14, 2004. Accepted February 2, 2005.Address requests for reprints to: Markus M. Lerch, M.D., Department of

astroenterology, Endocrinology and Nutrition, Ernst-Moritz-Arndt-Univer-ität Greifswald, Friedrich-Loeffler-Strasse 23a, 17487 Greifswald, Ger-any. e-mail: [email protected]; fax: (49) 3834 867234.Supported by the Deutsche Forschungsgemeinschaft (to M.M.L. and

.K.), the BMBF (IZKF Münster to J.S. and M.M.L), and the Deutscherebshilfe Mildred-Scheel-Stiftung (to M.M.L. and J.M.).The authors thank Dr. Elmar Wachter for his support in protein

equencing and C. Westermann, S. Agyemang, and U. Breite for expert
echnical assistance.

extracellular cleavage of e-cadherin by leukocyte elastase ... · extracellular cleavage of...

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