April 2000

2206THERAPEUTIC APPLICATION OF INTERLEUKIN-IB-CON­VERTING ENZYME (ICE) INHffilTOR DECREASES MORTAL­ITY OF SEVERE ACUTE EXPERIMENTAL PANCREATITIS.Bettina Rau, Adam S. Paszkowski, Jens M. Mayer, Hans G. Beger, Vnivof VIm, Vim, Germany.

There are several lines of evidence that cytokines activated by ICE play aninstrumental role in the course of acute pancreatitis (AP). Recent studieshave shown beneficial effects on local pancreatic damage and survival ofsevere AP in rats pretreated with an ICE-inhibitor as well as in ICE-knock­out mice. Hence, no study has ever assessed the effect of ICE-inhibitionadministered after the onset of the disease, which comes closer to theclinical setting. Material and Methods. Severe AP was induced by retro­grade, pressure-controlled infusion of 5% sodium-taurocholate in 45 maleWistar-rats, A new, highly selective, competitive and irreversible inhibitorof ICE (Bachem, Germany) was applied intraperitoneally (i.p.) at a dosageof 0,25mg every 12 hrs. Control animals of group I (n=18) were treatedi.p. with saline 6h after induction of AP, rats of group II (n= 13) receivedtreatment with ICE-inhibitor 6h, rats of group III (n= 14) 12h after onset ofAP. Following an observation period of 7 days surviving rats were sacri­ficed and serum, pancreas and lung were used for subsequent analysis,values are shown as mean j: SEM. Results. Treatment with ICE inhibitordecreased 7d-mortality from 88,9% (16/18) in controls to 46, I% (6/13,p=0,02) in group II and to 42,9% (6/14, p=0,008) in group III. Morpho­metric analysis of the pancreas revealed 0% remaining viable acinar cellsin group I, 22% j: 8 in group II, and 34% ± 9 in group III, whereas themean extent of necrosis was 78% ± 2,47% ± 12 and 47% ± 7, respec­tively (p=n.s.). Morphometry of the lungs did not show any differences interms of pulmonary damage. MPO concentrations (Ulg protein) in pancreasand lungs were as follows: 2482 ± 87 and 791 z 93 in group I, 456 ± 288and 356 ± 252 in group II, and 6 ± I and 104 j: 38 in group III,respectively (p=n.s.). There were no significant changes of amylase, lipaseand LDH levels between the surviving animals of the three groups. Con­clusion. Our results demonstrate that inhibition of ICE significantly im­proves survival in severe AP. This may be due to decreased neutrophilmediated tissue injury. Moreover, inhibition is still effective after a ther­apeutic window of 12 hours. Based on the current findings future studies onthe clinical application of ICE-inhibiting substances in AP seem to bepromising.

2207IMPAIRED RECRUITMENT OF HEPATOCYTE VESICLE ASSO­CIATED MEMBRANE PROTEIN (VAMP) BY FEEDING INCHRONIC PANCREATITIS (CP).Kumudesh C. Sritharan, Gudrun Aspelund, Lori A. Slezak, Bhanu P. Jena,Dana K. Andersen, Yale Vniv Sch of Medicine, New Haven, CT.

CP is accompanied by altered hepatocyte endocytosis. This may resultfrom altered vesicle trafficking which affects not only glucose transport butalso other transport processes. VAMP along with syntaxin and SNAP-25are proteins, termed SNARES, which mediate vesicle docking and fusionat the cell plasma membrane. Immunolocalization of VAMP in subcellularfractions of hepatocytes was determined in sham operated rats and rats inwhom CP had been induced 2-3 months earlier by pancreatic duct oleicacid infusion. After 45 minutes of duodenal intubation alone (fasting) orintubation plus duodenal feeding, livers were removed, homogenized andultracentrifuged, and microsomal pellets were separated by sucrose densitygradient ultracentrifugation. VAMP in plasma membrane (PM) and golgi(G) fractions were determined by western blot and scanning densitometry.The mean (±SEM) PM:G ratio of VAMP increased from 0.6 ± .13 infasting sham livers to 1.2 ± .37 in fed livers (n = 6,6; p<.05 by WilcoxonSigned Rank Test). However, there was no change in the PM:G ratio ofVAMP in CP livers after duodenal feeding (1.2 ::!: .25 vs. 1.0 ::!: .19 n =6, 6; P = NS). Fluorescence microscopy of 4% paraformaldehyde fixedtissue sectioned and stained with monoclonal VAMP Ab confirmed ourfindings. We conclude that feeding normally induces a shift in VAMP fromthe golgi compartment to the plasma membrane, but the presence of VAMPat the PM is diminished in livers from rats with CPo This effect may playa role in the altered hepatocyte vesicle transport associated with CP.

AGAA429

2208

FUNCTIONAL IMPAIRMENT OF PERITONEAL MACROPHAGE(M(J) IN EXPERIMENTAL SEVERE ACUTE PANCREATITIS INRATS.Yoshifumi Takeyama, Yuichi Hori, Nozomi Veno, Takashi Veda, Yo­shikazu Kuroda, Kobe Vniv Sch of Med, Kobe, Japan.

Background and Aim: In rat caerulein-induced pancreatitis, which is anexperimental model for edematous pancreatitis, we have found that theM<f>s are at least primed for further stimulation, such as lipopolysaccharide(LPS), in lung, liver (Kupffer cells), and peritoneal cavity within 24 hoursafter administration of caeruJein. We have also found expression of induc­ible nitric oxide synthase (NOS) mRNA in peritoneal M<f>s 12 hous afteradministration of caeruJein. These results indicate that resident M<f>s areprimed systemically in the early stage of mild pancreatitis. The aim of thisstudy is to clarify the status of peritoneal M<f>s in severe acute pancreatitis.Methods: Severe necrotizing pancreatitis was developed in male Fisher ratsby the retrograde injection of 0.1 ml of 5% deoxycholate into the bilio­pancreatic duct. After 5 hours, peritoneal M<f>s were collected by lavageand culture, and their functional status was estimated in vitro. Intracellularfree radical production was measured by nitro blue tetrazolium methodwith or without phorbol ester stimulation (20JLM phorbol myristate acetate). Nitric oxide (NO) production upon LPS stimulation was measured byGries' method. Phagocytotic activity were estimated by uptake of fluores­cent microspheres, fluoresbrite™(0.75JLm). Results: In the peritoneal M<f>scollected from the rat with pancreatitis, the intracellular free radical pro­duction without or with phorbol ester stimulation was depressed to 80% or69% of that in the peritoneal M<f>s from the rats with sham operation,respectively. The peritoneal M<f>s did not respond to phorbol ester stimu­lation at all. Moreover, in the peritoneal M<f>s from the rats with pancre­atitis, NO production upon in vitro LPS stimulation (loong/ml) and phago­cytotic activity were inhibited to 44% and 57% of those in the peritonealM<f>s from the rats with sham operation, respectively. Conclusion andDiscussion: Peritoneal M<f>s are shown to be functionally impaired in theearly stage of the experimental severe acute pancreatitis, in contrast that theresident M<f>s including peritoneal M<f>s are at least primed in experimentalmild pancreatitis. This phenomenon possibly correlates with the differ­ences in systemic involvement and prognosis between mild and severepancreatitis.

2209

EXPERIMENTAL PANCREATITIS IN KERATIN-NULL MICEREVEALS MAJOR DIFFERENCES IN PANCREATIC ACINARCELL AND HEPATOCYTE KERATIN CYTOPROTECTIVEFUNCTIONS.Diana M. Toivola, Helene Baribault, Thomas Magin, Bishr Omary, PaloAlto VA Med Ctr, Palo Alto, CA; Deltagen, San Carlos, CA; Vniv ofBonn, Bonn, Germany.

Background: Intermediate filaments of exocrine pancreatic acinar cellsreportedly consist of keratin heterodimers (K8 and K18), which form twodistinct pools: a cytoplasmic pool and a plasma membrane-proximal apico­lateral cellular pool. Previous studies showed that hepatocyte K8/18 fila­ments playa major role in providing mechanical integrity and protectionfrom environmental stresses, while disruption of pancreatic acinar cellcytoplasmic filaments in transgenic mice expressing KI8 arg89-->cys(KI8C) had no significant effect. This suggested that acinar cell apico­lateral filaments may be important in carrying out these cytoprotectiveroles, or that K81l8 serve different functions in hepatocytes and acinarcells. Aim: To use K8 and Kl8 null mice to determine if pancreatic keratinsprovide a cytoprotective function that is similar to the important functionthat they provide in hepatocytes. Methods: Keratins were assessed inpancreata of wild type, K8 and K18 null mice and heterozygous mice usingconfocal and immune electron microscopy, and biochemically. Caeruleininjections or a choline-deficient methionine supplemented diet were used toinduce experimental acute pancreatitis. We assessed pancreatitis histolog­ically and serologically, and acini were isolated to examine their secretoryfunctions and viability. Results: Biochemical and immunofluorescencestaining showed that K8 null mice lack both keratin pools while, unex­pectedly, KI8 null mice lack only the cytoplasmic keratin pool. Contraryto earlier reports, acinar cells also express Kl9 which explains the presenceof apico-lateral keratins in K18 null mice. K8 null mice tolerated diet- andcaerulein-induced pancreatitis well, as determined histologically and byserologic testing. Also, acini isolated from K8 null mice had a similarviability and yield as compared to acini that lack cytoplasmic filamentsfrom KI8 null or KI8C mice. Conclusions: KI9 is a normal constituent ofacinar cell intermediate filaments, and acinar cell and hepatocyte K8/18differ significantly in the extent of their participation in cytoprotectivefunctions. This difference may be represented by non-keratin exocrinepancreatic proteins that provide keratin equivalent functional redundancy.