How and Where Does Acute Pancreatitis
Begin?
Michael L. Steer, MD \s=b\ Circumstantial
evidence suggests that gallstone-induced pancreatitis triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the dietinduced, secretagogue-induced, and duct obstruction\p=n-\ induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lyososomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells. is
(Arch Surg. 1992;127:1350-1353)
gallstone-induced pancreatitis is precipitated by the passage of biliary into through the Acute terminal believed that bile duct. It is a
i
tract stone
or
generally pancreatitis results from digestive injury of the gland by enzymes that it normally synthesizes and secretes. Evi¬ dence leading to this conclusion includes the following: (1) The pancreas synthesizes a large number and amount of digestive enzymes, which, if activated, could potentially injure the gland. (2) Activated digestive enzymes have been detected within the pancreas during acute pancreati¬ tis.1 (3) The morphologic appearance of acute pancreatitis resembles that of digestive necrosis. The mechanism(s) by which stone passage might trigger pancreatitis, however, has not been defined. Three theories have been proposed to explain this association. The first, the so-called commonchannel theory,2 suggested that the offending stone could obstruct the biliopancreatic ductal system, creating a com¬ mon channel behind it through which bile might pass, re¬ common
flux into the pancreatic duct, and trigger pancreatitis. This theory has been criticized because (1) pancreatic secretory pressure usually exceeds bile secretory pressure, and bile
Accepted for publication April 12, 1992. From the Department of Surgery, Beth Israel Hospital
and Harvard Medical School, Boston, Mass. Presented at the Scientific Symposium in Honor of Dr William Silen,
Boston, Mass, May 16, 1991. Reprint requests to the Department of Surgery, Beth Israel Hospital, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215 (Dr Steer).
unlikely to pass into the pancreatic ductal system through a common channel3; (2) bile experimen¬ tally perfused into the pancreatic ductal system does not cause pancreatitis4; and (3) not all patients with gallstoneinduced pancreatitis have a common biliopancreatic duc¬ tal system that is long enough to result in such a common
would thus be
channel after distal obstruction. The second theory proposed that the offending stone might pass through and stretch the sphincter of Oddi, making it an incompetent barrier to reflux of duodenal juices containing activated digestive enzymes.5 Because surgical or endoscopie sphincterotomy does not routinely lead to pancreatitis, this theory is an unlikely explanation for the pathogenesis of pancreatitis. The third and, by exclusion, most likely explanation is that the offending stone obstructs the pancreatic duct and that, with continued secretion into the closed pancreatic ductal space, ductal hypertension ensues. Some investiga¬ tors have suggested that this leads to ductal rupture and extravasation of pancreatic juice into the gland paren¬ chyma. However, this must be an incomplete explanation because pancreatic juices generally contain only inactive proteolytic enzyme zymogens (eg, trypsinogen, chymotrypsinogen, and procarboxypeptidase). During the past decade, my colleagues and I have tried to explore the events that might link stone passage to the development of acute pancreatitis. In our studies, we have exploited several models of pancreatitis that can be in¬ duced in animals. We have focused our studies on the cell biologic processes of enzyme synthesis, intracellular trans¬ port, sorting, and secretion. In these studies, we have sought events that might be common to all the models of pancreatitis in the belief that such common features would also be features of clinical pancreatitis. METHODS
Experimental Models Diet-induced pancreatitis can be established by feeding young female mice a choline-deficient, ethionine-supplemented (CDE) diet.6 If the diet is continuously administered, all of the mice die within 5 days of acute hemorrhagic pancreatic necrosis. We modified this protocol by administering the diet for only 24 hours and evaluated pancreatic function 48 to 72 hours after the initi¬ ation of the CDE diet, at a time when gross and light microscopic evidence of pancreatic injury is not present. This protocol results in a 5-day mortality rate of 50% to 60%. Secretagogue-induced pancreatitis can be established by ad¬ ministering a dose of the cholecystokinin analogue caerulein in excess of that which stimulates a maximal rate of pancreatic pro-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
of cathepsin B during secretagogueinduced pancreatitis. Rats were infused with saline (shaded bars) or sa¬ line containing caerulein at a concentration designed to deliver either 0.25 u.g-/cg~'-rr' (solid bars) or 0.5 \ig-kg~'1 h''1 (hatched bars) for 3.5 hours before death, and the pancreas was subcellularly fractioned. Cathepsin B activity, measured in each fraction, is expressed as a per¬ cent ofthe total found in postnuclear homogenate. 1.3 KP indicates 1300 g/15-minute pellet (primarily zymogen granules); 12 KP, 12 000 g/12minute pellet (primarily lysosomes and mitochondria); 105 KP, 105 000 g/60-minute pellet (primarily microsomes); and 105 KS, 105 000 g/60minute supernatant (soluble fraction). Results represent mean values, and vertical bars represent SEs obtained from four or more separate fractionations, each performed with the use of samples from different animals (from Saluja et aP°).
Fig 2.—Subcellular redistribution
Fig 1.—Amino acid uptake, protein synthesis, and secretion during secretagogue-induced pancreatitis. Rats were preinfused with saline alone (solid circles)
or
saline
containing sufficient caerulein
to
deliver
¡ig/kg~'/h~' (open circles) for 1 hour and were then given a pulse of tritiated phenylalanine followed by a bolus of nonradioactive phenyl-
5
alanine; the infusion was continued for varying times. At selected inter¬ vals, rats (n=7) in each group were killed, and trichloroacetic acid-
was measured in pancreas homogenate. Asterisks indicate values in caerulein-infused animals that were signif¬ icantly different from those found in saline-infused rats (from Saluja et
precipitable radioactivity aPV-
tein secretion. When given by intravenous infusion to rats, supramaximal stimulation with caerulein results in a reversible form of interstitial edematous pancreatitis that develops within 1 hour of the onset of caerulein infusion.7 Duct obstruction in rabbits does not cause morphologic evi¬ dence of pancreatic injury or inflammation but eventually leads to atrophy of the exocrine pancreas. We induced duct obstruction by cannulating the rabbit pancreatic duct and elevating the out¬ flow portion of the cannula to a vertical position such that obstruction occurred when the hydrostatic pressure of the se¬ creted juice reached the secretory pressure of the exocrine pancreas.8 While this model does not lead to pancreatic injury in rabbits, we believe that it mimics the effect of an obstructing bil¬ iary tract stone in humans. Duct obstruction in opossums leads to acute hemorrhagic pan¬ creatitis, with a 14-day mortality rate of 100%.91° We have exam¬ ined the early events that follow opossum pancreatic duct obstruction with the goal of determining whether acute pancre¬ atitis begins in acinar cells or, as suggested by other investigators, in either the periductal" or perilobular areas.12
Hyperamylasemia and
Pancreatic Edema In each of the models described above, a marked increase in
amylase level and in the pancreatic water content can be easily demonstrated.813"15 These changes appear within 2 days of serum
CDE diet administration and within hours of either duct obstruc¬ tion or supramaximal secretagogue stimulation.
Amino Acid
Uptake and
Protein
Synthesis
The pancreatic acinar cell is, on a cell-to-cell basis, the most ac¬ tive protein-synthesizing cell in the body, and more than 90% of newly synthesized protein in the pancreas is digestive enzyme protein. This observation suggested the possibility that acute
pancreatitis might involve an abnormality in either amino acid uptake or protein synthesis by the pancreas. We examined these processes (Fig 1) in each of the models but found that in each, amino acid uptake and protein synthesis were unaltered.813"15
Digestive Enzyme and Lysosomal Hydrolase Segregation Digestive enzyme zymogens and lysosomal hydrolases are synthesized on the rough endoplasmic reticulum and transported to the Golgi complex. As they traverse the Golgi stacks, lysoso¬ mal hydrolases are glycosylated and phosphorylated at the 6-position
of
mannose
residues. As
a
result, the mannose-6-
phosphorylated lysosomal hydrolases are bound to mannose-6phosphate-specific receptors that facilitate their transport from the Golgi complex to the prelysosomal compartment, where, as a result of the acidic environment, the hydrolases dissociate from mannose-6-phosphate receptors, allowing those receptors to shuttle back to the Golgi complex.16 In contrast to lysosomal hy¬ drolases, digestive zymogens and other exported proteins are not mannose-6-phosphorylated and, as a result, are not bound to mannose-6-phosphate receptors. Rather, they pass through the Golgi complex, are packaged in condensing vacuoles that mature into zymogen granules, and are discharged at the luminal cell surface as a result of fusion-fission of the zymogen granule mem¬
brane with the luminal plasmalemma.17 The lysosomal hydrolase cathepsin B can activate trypsinogen,18,19 and trypsin can activate the remaining zymogens. Based on this observation, we suspected that premature activation of di¬ gestive zymogens in pancreatitis could be the result of colocalization of digestive zymogens with lysosomal hydrolases, such as cathepsin B. We utilized two techniques to evaluate the possibility that colocalization of digestive zymogens with lysosomal hydro¬ lases might occur during pancreatitis. The first involves differen¬ tial centrifugation of samples obtained from the homogen¬ ized pancreas and assay of the various subcellular fractions to determine the location of digestive zymogens and lyso¬ somal hydrolases during the early stages of experimental pancre¬ atitis. The second approach involves either light or electron mi¬ croscopic immunolocalization of digestive zymogens and lysos-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
Fig 3.—Colocalization of digestive enzyme zymogens and lysosomal hydrolase during secretagogue-induced pancreatitis. Photographs show indi¬ rect immunofluorescence of pancreatic acinar cells of rats infused with caerulein for 1 hour and illustrate immunolabeling obtained with antizymogen (A) and anticathepsin D (B) serum samples in corresponding fields from two semithin (1-ix.m) sections cut next to each other from same block. An¬ tizymogen immunolabeling appears diffuse throughout the cytoplasm with heavy reaction of zymogen granules (localized at cell apex) and, espe¬ cially, on large, heterogeneous granules localized preferentially in the Golgi area. Anticathepsin labeling is restricted to vacuoles with localization in the Golgi area. Numbers (1 through 10) indicate vacuoles in two adjacent sections that were labeled by both serum samples used (original mag¬ nification
X
1200) (from Watanabe
et
aPV.
omal hydrolases with the use of either fluorescein-labeled or gold particle-labeled antibodies directed at each of these types of
proteins.
We found that the lysosomal hydrolase cathepsin B moves from the lysosome-enriched subcellular fraction to the heavier zy¬ mogen granule-enriched fraction during the early stages of dietinduced and secretagogue-induced (Fig 2) pancreatitis and shortly after rabbit pancreatic duct obstruction.82"21 With the use of immunolocalization techniques, lysosomal hydrolases were found to be colocalized with digestive zymogens within large cytoplasmic vacuoles in both diet-induced and secretagogueinduced (Fig 3) pancreatitis.'^17-2"22 Colocalization of lysosomal hydrolases with digestive zymogens was also noted after rabbit pancreatic duct obstruction.817 In each of these three models, colocalization of digestive zymogens with lysosomal hydrolases was associated with an increased fragility of the organelles con¬
taining lysosomal hydrolases.
Digestive Enzyme Secretion We evaluated the process of digestive enzyme secretion during the early stages of diet-induced and secretagogue-induced pan¬ creatitis and shortly after pancreatic duct obstruction. In each of these three conditions, digestive enzyme secretion into the ductal space was markedly reduced or abolished.81314 The intracellular events that lead to this inhibition of secretion appear to differ in the three conditions. Thus, after CDE diet administration, there is a defect in receptor-mediated stimulus-secretion coupling that appears to involve the G-protein coupled to phospholipase C.23,24 In secretagogue-induced pancreatitis, interaction with lowaffinity inhibitory cholecystokinin receptors25 and the possible involvement of a recently identified plasma factor, which is expressed after supramaximal stimulation with caerulein, maybe responsible for the inhibition of digestive enzyme secretion.26 The mechanisms responsible for inhibition of secretion after duct ob¬ struction have not been identified.
Site of
Origin
Considerable controversy has surrounded the question of where acute pancreatitis begins. As noted previously, some
investigators have suggested that ductal disruption and extrava¬ sation of pancreatic juice into the parenchyma of the gland is an early event and that the early inflammatory changes occur in the periductal areas of the pancreas." Others have noted that the perilobular fat necrosis is an early finding in many cases of pan¬ creatitis and, as a result, have suggested that the earliest inflam¬ matory changes occur in the peripancreatic tissue.12 To study this issue, we evaluated the morphologic changes that characterize the first 24 hours after ligation of the opossum pancreatic duct.27 As noted by other investigators, ligation of the opossum pancreatic duct leads to hemorrhagic pancreatitis, which is evident within 3 days of duct ligation and fatal within 14 days of duct ligation.11'1" We noted evidence of acinar cell injury within 3 hours of duct li¬ gation, a time when no evidence of peripancreatic or periductal injury could be detected. As a result, we concluded that the early events in acute pancreatitis most likely involve the acinar cells of the pancreas and that peripancreatic as well as periductal inflam¬ mation
occurs
later.
OVERVIEW AND CONCLUSIONS overall sense, the previously described studies suggested that the following sequence of events may underlie the development of acute pancreatitis. The initi¬ ating stimulus appears to be stone-induced pancreatic duct obstruction, which leads to ductal hypertension. Ductal hypertension, by an as yet unclear mechanism, results in the inhibition of digestive enzyme secretion and in the colocalization of digestive zymogens with lysosomal hydro¬ lases inside acinar cells. This colocalization phenomenon could, theoretically, result in digestive enzyme activation and leakage of activated digestive enzymes into the acinar cell cytoplasmic space. The final effect of this process could, we suggest, be acinar cell injury and acute pancre¬ atitis. This hypothesis, if valid, may redirect our approach to the treatment and prevention of pancreatitis. For example, the value of interventions that inhibit pancreatic exocrine In have
an
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
secretion in the management of acute pancreatitis would seem to be minimal if, in fact, an early event in the initi¬ ation of pancreatitis is a blockade of secretion. Rather, in¬ terventions that restore secretion may be of greater bene¬ fit. These findings would also suggest that to be effective, therapies for acute pancreatitis must be directed at events occurring within acinar cells rather than in the ducts or
elsewhere.
Obviously, a number of unanswered questions remain regarding the pathogenesis of acute pancreatitis. One of
the most important of these is the question of what deter¬ mines the ultimate severity of acute pancreatitis. The events described herein are noted during diet-induced
pancreatitis, during secretagogue-induced pancreatitis, and after rabbit pancreatic duct obstruction, yet the out¬ comes of these three models are remarkedly different. The CDE diet induces lethal hemorrhagic pancreatitis, while supramaximal secretagogue stimulation causes only re¬ versible interstitial pancreatic edema. Rabbit pancreatic duct obstruction leads only to exocrine pancreatic atrophy. Thus, there must be other, as yet unidentified, events that, when coupled with those described herein, determine the
ultimate severity of pancreatitis. The identification of those events will require the efforts of many investigators and, most likely, at least another decade of studies. This work tutes of
was
supported by grant 31396 from
the National Insti¬
Health, Bethesda, Md.
The author is indebted to his many coworkers, especially Jacopo Meldolesi, MD, and Ashok Saluja, PhD, whose contributions to this work were critical. References 1. Geokas MD, Rinderknecht H. Free proteolytic enzymes in pancreatic juice of patients with acute pancreatitis. Am J Dig Dis. 1974;19:591-598. 2. Opie EL. The etiology of acute hemorrhagic pancreatitis. Bull Johns Hopkins Hosp. 1901;12:182-192. 3. Menguy RB, Hallenbeck GA, Bollman JL, Grindlay JH. Intraductal pressures and sphincteric resistance in canine pancreatic and biliary ducts after various stimuli. Surg Gynecol Obstet. 1958;106:306-320. 4. Robinson TM, Dunphy JE. Continuous perfusion of bile protease activators through the pancreas. JAMA. 1963;183:530-533. 5. McCutcheon AD, Race D. Experimental pancreatitis: a possible etiology of post-operative pancreatitis. Ann Surg. 1962;155:523-531. 6. Lombardi B, Estes LW, Longnecker DS. Acute hemorrhagic pancreatitis
(massive necrosis) with fat necrosis induced in mice by DL-ethione fed with choline-deficient diet. Am J Pathol. 1975;79:465-480. 7. Lampel M, Kern H. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch A Pathol Anat Histopathol. 1977;373:107-117. 8. Saluja A, Saluja M, Villa A, Rutledge P, Meldolesi J, Steer M. Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization. J Clin Invest. 1989;84:1260-1266. a
9. Senninger N, Moody FG, Van Buren DH, Coelho JCU, Li YF. Effect of biliary obstruction on pancreatic exocrine secretion in conscious opossums. Surg Forum. 1984;35:226-228. 10. Cavuoti OP, Moody FG, Martinez G. Role of pancreatic duct occlusion with prolamine (Ethibloc) in necrotizing pancreatitis. Surgery. 1988; 103:261-366. 11. Foulis AK.
Histological evidence of initiating factors in acute necrotizing pancreatitis in man. J Clin Pathol. 1980;33:1123-1131. 12. Kloeppel G, Dreyer T, Willemer S, Kern HF, Adler G. Human acute pancreatitis: its pathogenesis in the light of immunocytochemical and ultrastructural findings of acinar cells. Virchows Arch A Pathol Anat Histopathol.
1986;409:791-803. 13. Gilliland L, Steer ML. Effects of ethionine on digestive enzyme synthesis and discharge by mouse pancreas. Am J Pathol. 1980;79:465\x=req-\
476. 14.
Saluja AK, Saito I, Saluja M, et al. In-vivo rat pancreatic acinar cell during supramaximal stimulation with caerulein. Am J Physiol.
function
1985;249:G702-G710. 15. Watanabe O, Baccino FM, Steer ML, Meldolesi J. Effects of supramaximal caerulein stimulation of the ultrastructure or rat pancreatic acinar cell:
early morphological changes during the development of experimental pancreatitis. Am J Physiol. 1984;246:G457-G467. 16. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease J Clin Invest. 1986;77:1-6. 17. Palade GE. Intracellular aspects of the process of
states.
protein secretion. Science. 1975;189:347-358. 18. Greenbaum LA, Hirshkowitz A. Endogenous cathepsin activates trypsinogen in extracts of dog pancreas. Proc Soc Exp Biol Med. 1961;107: 74-76. 19. Figarella C, Miszczuk-Jamska B, Barrett AJ. Possible lyosomal activation of pancreatic zymogens: activation of both human trypsinogens by cathepsin B and spontaneous acid activation of human trypsinogen 1. Biol Chem Hoppe Seyler. 1988;369(suppl):293-298. 20. Saluja AK, Hashimoto S, Saluja M, Powers RE, Meldolesi J, Steer ML. Subcellular redistribution of lysosomal enzymes during caerulein-induced Am J Physiol. 1987;251:G508-G516. 21. Ohshio G, Saluja A, Leli U, Sengupta A, Steer ML. Esterase inhibitors prevent lysosomal enzyme redistribution in two non-invasive models of ex-
pancreatitis.
perimental pancreatitis. Gastroenterology. 1989;96:853-859. 22. Saito I, Hashimoto S, Saluja A, Steer ML, Meldolesi J. Intracellular transport of pancreatic zymogens during caerulein supramaximal stimulation. Am J Physiol. 1987;251:G517-G526. 23. Powers RE, Saluja AK, Houlihan MJ, Steer ML. Diminished agonist\x=req-\ stimulated inositol triphosphate generation blocks stimulus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreati-
tis. J Clin Invest. 1986;77:1668-1674. 24. Leli U, Saluja A, Picard L, Zavertnik A, Steer ML. Effects of a choline\x=req-\ deficient ethionine-supplemented diet on phospholipase C activity in mouse pancreatic acinar cell membranes and in electropermeabilized mouse pancreatic acini. J Pharmacol Exp Ther. 1990;253:847-850. 25. Saluja A, Saluja M, Printz H, Zavertnik A, Sengupta A, Steer ML. Experimental pancreatitis is mediated by low affinity CCK receptors which inhibit digestive enzyme secretion. Proc Natl Acad Sci U S A.1989;86:8969\x=req-\ 8971. 26. Saluja M, Saluja A, Lerch MM, Steer M. A plasma factor which is expressed during supramaximal stimulation causes in-vitro subcellular redistribution of lysosomal enzymes in rat exocrine pancreas. J Clin Invest. 1991; 87:1280-1285. 27. Lerch MM, Saluja A, Ramarao P, et al. Common pancreatic-bile duct (P-BD) obstruction in the opossum causes the early development of necrotizing pancreatitis. Pancreas. 1990;5:719.
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
Begin?
Michael L. Steer, MD \s=b\ Circumstantial
evidence suggests that gallstone-induced pancreatitis triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the dietinduced, secretagogue-induced, and duct obstruction\p=n-\ induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lyososomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells. is
(Arch Surg. 1992;127:1350-1353)
gallstone-induced pancreatitis is precipitated by the passage of biliary into through the Acute terminal believed that bile duct. It is a
i
tract stone
or
generally pancreatitis results from digestive injury of the gland by enzymes that it normally synthesizes and secretes. Evi¬ dence leading to this conclusion includes the following: (1) The pancreas synthesizes a large number and amount of digestive enzymes, which, if activated, could potentially injure the gland. (2) Activated digestive enzymes have been detected within the pancreas during acute pancreati¬ tis.1 (3) The morphologic appearance of acute pancreatitis resembles that of digestive necrosis. The mechanism(s) by which stone passage might trigger pancreatitis, however, has not been defined. Three theories have been proposed to explain this association. The first, the so-called commonchannel theory,2 suggested that the offending stone could obstruct the biliopancreatic ductal system, creating a com¬ mon channel behind it through which bile might pass, re¬ common
flux into the pancreatic duct, and trigger pancreatitis. This theory has been criticized because (1) pancreatic secretory pressure usually exceeds bile secretory pressure, and bile
Accepted for publication April 12, 1992. From the Department of Surgery, Beth Israel Hospital
and Harvard Medical School, Boston, Mass. Presented at the Scientific Symposium in Honor of Dr William Silen,
Boston, Mass, May 16, 1991. Reprint requests to the Department of Surgery, Beth Israel Hospital, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215 (Dr Steer).
unlikely to pass into the pancreatic ductal system through a common channel3; (2) bile experimen¬ tally perfused into the pancreatic ductal system does not cause pancreatitis4; and (3) not all patients with gallstoneinduced pancreatitis have a common biliopancreatic duc¬ tal system that is long enough to result in such a common
would thus be
channel after distal obstruction. The second theory proposed that the offending stone might pass through and stretch the sphincter of Oddi, making it an incompetent barrier to reflux of duodenal juices containing activated digestive enzymes.5 Because surgical or endoscopie sphincterotomy does not routinely lead to pancreatitis, this theory is an unlikely explanation for the pathogenesis of pancreatitis. The third and, by exclusion, most likely explanation is that the offending stone obstructs the pancreatic duct and that, with continued secretion into the closed pancreatic ductal space, ductal hypertension ensues. Some investiga¬ tors have suggested that this leads to ductal rupture and extravasation of pancreatic juice into the gland paren¬ chyma. However, this must be an incomplete explanation because pancreatic juices generally contain only inactive proteolytic enzyme zymogens (eg, trypsinogen, chymotrypsinogen, and procarboxypeptidase). During the past decade, my colleagues and I have tried to explore the events that might link stone passage to the development of acute pancreatitis. In our studies, we have exploited several models of pancreatitis that can be in¬ duced in animals. We have focused our studies on the cell biologic processes of enzyme synthesis, intracellular trans¬ port, sorting, and secretion. In these studies, we have sought events that might be common to all the models of pancreatitis in the belief that such common features would also be features of clinical pancreatitis. METHODS
Experimental Models Diet-induced pancreatitis can be established by feeding young female mice a choline-deficient, ethionine-supplemented (CDE) diet.6 If the diet is continuously administered, all of the mice die within 5 days of acute hemorrhagic pancreatic necrosis. We modified this protocol by administering the diet for only 24 hours and evaluated pancreatic function 48 to 72 hours after the initi¬ ation of the CDE diet, at a time when gross and light microscopic evidence of pancreatic injury is not present. This protocol results in a 5-day mortality rate of 50% to 60%. Secretagogue-induced pancreatitis can be established by ad¬ ministering a dose of the cholecystokinin analogue caerulein in excess of that which stimulates a maximal rate of pancreatic pro-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
of cathepsin B during secretagogueinduced pancreatitis. Rats were infused with saline (shaded bars) or sa¬ line containing caerulein at a concentration designed to deliver either 0.25 u.g-/cg~'-rr' (solid bars) or 0.5 \ig-kg~'1 h''1 (hatched bars) for 3.5 hours before death, and the pancreas was subcellularly fractioned. Cathepsin B activity, measured in each fraction, is expressed as a per¬ cent ofthe total found in postnuclear homogenate. 1.3 KP indicates 1300 g/15-minute pellet (primarily zymogen granules); 12 KP, 12 000 g/12minute pellet (primarily lysosomes and mitochondria); 105 KP, 105 000 g/60-minute pellet (primarily microsomes); and 105 KS, 105 000 g/60minute supernatant (soluble fraction). Results represent mean values, and vertical bars represent SEs obtained from four or more separate fractionations, each performed with the use of samples from different animals (from Saluja et aP°).
Fig 2.—Subcellular redistribution
Fig 1.—Amino acid uptake, protein synthesis, and secretion during secretagogue-induced pancreatitis. Rats were preinfused with saline alone (solid circles)
or
saline
containing sufficient caerulein
to
deliver
¡ig/kg~'/h~' (open circles) for 1 hour and were then given a pulse of tritiated phenylalanine followed by a bolus of nonradioactive phenyl-
5
alanine; the infusion was continued for varying times. At selected inter¬ vals, rats (n=7) in each group were killed, and trichloroacetic acid-
was measured in pancreas homogenate. Asterisks indicate values in caerulein-infused animals that were signif¬ icantly different from those found in saline-infused rats (from Saluja et
precipitable radioactivity aPV-
tein secretion. When given by intravenous infusion to rats, supramaximal stimulation with caerulein results in a reversible form of interstitial edematous pancreatitis that develops within 1 hour of the onset of caerulein infusion.7 Duct obstruction in rabbits does not cause morphologic evi¬ dence of pancreatic injury or inflammation but eventually leads to atrophy of the exocrine pancreas. We induced duct obstruction by cannulating the rabbit pancreatic duct and elevating the out¬ flow portion of the cannula to a vertical position such that obstruction occurred when the hydrostatic pressure of the se¬ creted juice reached the secretory pressure of the exocrine pancreas.8 While this model does not lead to pancreatic injury in rabbits, we believe that it mimics the effect of an obstructing bil¬ iary tract stone in humans. Duct obstruction in opossums leads to acute hemorrhagic pan¬ creatitis, with a 14-day mortality rate of 100%.91° We have exam¬ ined the early events that follow opossum pancreatic duct obstruction with the goal of determining whether acute pancre¬ atitis begins in acinar cells or, as suggested by other investigators, in either the periductal" or perilobular areas.12
Hyperamylasemia and
Pancreatic Edema In each of the models described above, a marked increase in
amylase level and in the pancreatic water content can be easily demonstrated.813"15 These changes appear within 2 days of serum
CDE diet administration and within hours of either duct obstruc¬ tion or supramaximal secretagogue stimulation.
Amino Acid
Uptake and
Protein
Synthesis
The pancreatic acinar cell is, on a cell-to-cell basis, the most ac¬ tive protein-synthesizing cell in the body, and more than 90% of newly synthesized protein in the pancreas is digestive enzyme protein. This observation suggested the possibility that acute
pancreatitis might involve an abnormality in either amino acid uptake or protein synthesis by the pancreas. We examined these processes (Fig 1) in each of the models but found that in each, amino acid uptake and protein synthesis were unaltered.813"15
Digestive Enzyme and Lysosomal Hydrolase Segregation Digestive enzyme zymogens and lysosomal hydrolases are synthesized on the rough endoplasmic reticulum and transported to the Golgi complex. As they traverse the Golgi stacks, lysoso¬ mal hydrolases are glycosylated and phosphorylated at the 6-position
of
mannose
residues. As
a
result, the mannose-6-
phosphorylated lysosomal hydrolases are bound to mannose-6phosphate-specific receptors that facilitate their transport from the Golgi complex to the prelysosomal compartment, where, as a result of the acidic environment, the hydrolases dissociate from mannose-6-phosphate receptors, allowing those receptors to shuttle back to the Golgi complex.16 In contrast to lysosomal hy¬ drolases, digestive zymogens and other exported proteins are not mannose-6-phosphorylated and, as a result, are not bound to mannose-6-phosphate receptors. Rather, they pass through the Golgi complex, are packaged in condensing vacuoles that mature into zymogen granules, and are discharged at the luminal cell surface as a result of fusion-fission of the zymogen granule mem¬
brane with the luminal plasmalemma.17 The lysosomal hydrolase cathepsin B can activate trypsinogen,18,19 and trypsin can activate the remaining zymogens. Based on this observation, we suspected that premature activation of di¬ gestive zymogens in pancreatitis could be the result of colocalization of digestive zymogens with lysosomal hydrolases, such as cathepsin B. We utilized two techniques to evaluate the possibility that colocalization of digestive zymogens with lysosomal hydro¬ lases might occur during pancreatitis. The first involves differen¬ tial centrifugation of samples obtained from the homogen¬ ized pancreas and assay of the various subcellular fractions to determine the location of digestive zymogens and lyso¬ somal hydrolases during the early stages of experimental pancre¬ atitis. The second approach involves either light or electron mi¬ croscopic immunolocalization of digestive zymogens and lysos-
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
Fig 3.—Colocalization of digestive enzyme zymogens and lysosomal hydrolase during secretagogue-induced pancreatitis. Photographs show indi¬ rect immunofluorescence of pancreatic acinar cells of rats infused with caerulein for 1 hour and illustrate immunolabeling obtained with antizymogen (A) and anticathepsin D (B) serum samples in corresponding fields from two semithin (1-ix.m) sections cut next to each other from same block. An¬ tizymogen immunolabeling appears diffuse throughout the cytoplasm with heavy reaction of zymogen granules (localized at cell apex) and, espe¬ cially, on large, heterogeneous granules localized preferentially in the Golgi area. Anticathepsin labeling is restricted to vacuoles with localization in the Golgi area. Numbers (1 through 10) indicate vacuoles in two adjacent sections that were labeled by both serum samples used (original mag¬ nification
X
1200) (from Watanabe
et
aPV.
omal hydrolases with the use of either fluorescein-labeled or gold particle-labeled antibodies directed at each of these types of
proteins.
We found that the lysosomal hydrolase cathepsin B moves from the lysosome-enriched subcellular fraction to the heavier zy¬ mogen granule-enriched fraction during the early stages of dietinduced and secretagogue-induced (Fig 2) pancreatitis and shortly after rabbit pancreatic duct obstruction.82"21 With the use of immunolocalization techniques, lysosomal hydrolases were found to be colocalized with digestive zymogens within large cytoplasmic vacuoles in both diet-induced and secretagogueinduced (Fig 3) pancreatitis.'^17-2"22 Colocalization of lysosomal hydrolases with digestive zymogens was also noted after rabbit pancreatic duct obstruction.817 In each of these three models, colocalization of digestive zymogens with lysosomal hydrolases was associated with an increased fragility of the organelles con¬
taining lysosomal hydrolases.
Digestive Enzyme Secretion We evaluated the process of digestive enzyme secretion during the early stages of diet-induced and secretagogue-induced pan¬ creatitis and shortly after pancreatic duct obstruction. In each of these three conditions, digestive enzyme secretion into the ductal space was markedly reduced or abolished.81314 The intracellular events that lead to this inhibition of secretion appear to differ in the three conditions. Thus, after CDE diet administration, there is a defect in receptor-mediated stimulus-secretion coupling that appears to involve the G-protein coupled to phospholipase C.23,24 In secretagogue-induced pancreatitis, interaction with lowaffinity inhibitory cholecystokinin receptors25 and the possible involvement of a recently identified plasma factor, which is expressed after supramaximal stimulation with caerulein, maybe responsible for the inhibition of digestive enzyme secretion.26 The mechanisms responsible for inhibition of secretion after duct ob¬ struction have not been identified.
Site of
Origin
Considerable controversy has surrounded the question of where acute pancreatitis begins. As noted previously, some
investigators have suggested that ductal disruption and extrava¬ sation of pancreatic juice into the parenchyma of the gland is an early event and that the early inflammatory changes occur in the periductal areas of the pancreas." Others have noted that the perilobular fat necrosis is an early finding in many cases of pan¬ creatitis and, as a result, have suggested that the earliest inflam¬ matory changes occur in the peripancreatic tissue.12 To study this issue, we evaluated the morphologic changes that characterize the first 24 hours after ligation of the opossum pancreatic duct.27 As noted by other investigators, ligation of the opossum pancreatic duct leads to hemorrhagic pancreatitis, which is evident within 3 days of duct ligation and fatal within 14 days of duct ligation.11'1" We noted evidence of acinar cell injury within 3 hours of duct li¬ gation, a time when no evidence of peripancreatic or periductal injury could be detected. As a result, we concluded that the early events in acute pancreatitis most likely involve the acinar cells of the pancreas and that peripancreatic as well as periductal inflam¬ mation
occurs
later.
OVERVIEW AND CONCLUSIONS overall sense, the previously described studies suggested that the following sequence of events may underlie the development of acute pancreatitis. The initi¬ ating stimulus appears to be stone-induced pancreatic duct obstruction, which leads to ductal hypertension. Ductal hypertension, by an as yet unclear mechanism, results in the inhibition of digestive enzyme secretion and in the colocalization of digestive zymogens with lysosomal hydro¬ lases inside acinar cells. This colocalization phenomenon could, theoretically, result in digestive enzyme activation and leakage of activated digestive enzymes into the acinar cell cytoplasmic space. The final effect of this process could, we suggest, be acinar cell injury and acute pancre¬ atitis. This hypothesis, if valid, may redirect our approach to the treatment and prevention of pancreatitis. For example, the value of interventions that inhibit pancreatic exocrine In have
an
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
secretion in the management of acute pancreatitis would seem to be minimal if, in fact, an early event in the initi¬ ation of pancreatitis is a blockade of secretion. Rather, in¬ terventions that restore secretion may be of greater bene¬ fit. These findings would also suggest that to be effective, therapies for acute pancreatitis must be directed at events occurring within acinar cells rather than in the ducts or
elsewhere.
Obviously, a number of unanswered questions remain regarding the pathogenesis of acute pancreatitis. One of
the most important of these is the question of what deter¬ mines the ultimate severity of acute pancreatitis. The events described herein are noted during diet-induced
pancreatitis, during secretagogue-induced pancreatitis, and after rabbit pancreatic duct obstruction, yet the out¬ comes of these three models are remarkedly different. The CDE diet induces lethal hemorrhagic pancreatitis, while supramaximal secretagogue stimulation causes only re¬ versible interstitial pancreatic edema. Rabbit pancreatic duct obstruction leads only to exocrine pancreatic atrophy. Thus, there must be other, as yet unidentified, events that, when coupled with those described herein, determine the
ultimate severity of pancreatitis. The identification of those events will require the efforts of many investigators and, most likely, at least another decade of studies. This work tutes of
was
supported by grant 31396 from
the National Insti¬
Health, Bethesda, Md.
The author is indebted to his many coworkers, especially Jacopo Meldolesi, MD, and Ashok Saluja, PhD, whose contributions to this work were critical. References 1. Geokas MD, Rinderknecht H. Free proteolytic enzymes in pancreatic juice of patients with acute pancreatitis. Am J Dig Dis. 1974;19:591-598. 2. Opie EL. The etiology of acute hemorrhagic pancreatitis. Bull Johns Hopkins Hosp. 1901;12:182-192. 3. Menguy RB, Hallenbeck GA, Bollman JL, Grindlay JH. Intraductal pressures and sphincteric resistance in canine pancreatic and biliary ducts after various stimuli. Surg Gynecol Obstet. 1958;106:306-320. 4. Robinson TM, Dunphy JE. Continuous perfusion of bile protease activators through the pancreas. JAMA. 1963;183:530-533. 5. McCutcheon AD, Race D. Experimental pancreatitis: a possible etiology of post-operative pancreatitis. Ann Surg. 1962;155:523-531. 6. Lombardi B, Estes LW, Longnecker DS. Acute hemorrhagic pancreatitis
(massive necrosis) with fat necrosis induced in mice by DL-ethione fed with choline-deficient diet. Am J Pathol. 1975;79:465-480. 7. Lampel M, Kern H. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch A Pathol Anat Histopathol. 1977;373:107-117. 8. Saluja A, Saluja M, Villa A, Rutledge P, Meldolesi J, Steer M. Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization. J Clin Invest. 1989;84:1260-1266. a
9. Senninger N, Moody FG, Van Buren DH, Coelho JCU, Li YF. Effect of biliary obstruction on pancreatic exocrine secretion in conscious opossums. Surg Forum. 1984;35:226-228. 10. Cavuoti OP, Moody FG, Martinez G. Role of pancreatic duct occlusion with prolamine (Ethibloc) in necrotizing pancreatitis. Surgery. 1988; 103:261-366. 11. Foulis AK.
Histological evidence of initiating factors in acute necrotizing pancreatitis in man. J Clin Pathol. 1980;33:1123-1131. 12. Kloeppel G, Dreyer T, Willemer S, Kern HF, Adler G. Human acute pancreatitis: its pathogenesis in the light of immunocytochemical and ultrastructural findings of acinar cells. Virchows Arch A Pathol Anat Histopathol.
1986;409:791-803. 13. Gilliland L, Steer ML. Effects of ethionine on digestive enzyme synthesis and discharge by mouse pancreas. Am J Pathol. 1980;79:465\x=req-\
476. 14.
Saluja AK, Saito I, Saluja M, et al. In-vivo rat pancreatic acinar cell during supramaximal stimulation with caerulein. Am J Physiol.
function
1985;249:G702-G710. 15. Watanabe O, Baccino FM, Steer ML, Meldolesi J. Effects of supramaximal caerulein stimulation of the ultrastructure or rat pancreatic acinar cell:
early morphological changes during the development of experimental pancreatitis. Am J Physiol. 1984;246:G457-G467. 16. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease J Clin Invest. 1986;77:1-6. 17. Palade GE. Intracellular aspects of the process of
states.
protein secretion. Science. 1975;189:347-358. 18. Greenbaum LA, Hirshkowitz A. Endogenous cathepsin activates trypsinogen in extracts of dog pancreas. Proc Soc Exp Biol Med. 1961;107: 74-76. 19. Figarella C, Miszczuk-Jamska B, Barrett AJ. Possible lyosomal activation of pancreatic zymogens: activation of both human trypsinogens by cathepsin B and spontaneous acid activation of human trypsinogen 1. Biol Chem Hoppe Seyler. 1988;369(suppl):293-298. 20. Saluja AK, Hashimoto S, Saluja M, Powers RE, Meldolesi J, Steer ML. Subcellular redistribution of lysosomal enzymes during caerulein-induced Am J Physiol. 1987;251:G508-G516. 21. Ohshio G, Saluja A, Leli U, Sengupta A, Steer ML. Esterase inhibitors prevent lysosomal enzyme redistribution in two non-invasive models of ex-
pancreatitis.
perimental pancreatitis. Gastroenterology. 1989;96:853-859. 22. Saito I, Hashimoto S, Saluja A, Steer ML, Meldolesi J. Intracellular transport of pancreatic zymogens during caerulein supramaximal stimulation. Am J Physiol. 1987;251:G517-G526. 23. Powers RE, Saluja AK, Houlihan MJ, Steer ML. Diminished agonist\x=req-\ stimulated inositol triphosphate generation blocks stimulus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreati-
tis. J Clin Invest. 1986;77:1668-1674. 24. Leli U, Saluja A, Picard L, Zavertnik A, Steer ML. Effects of a choline\x=req-\ deficient ethionine-supplemented diet on phospholipase C activity in mouse pancreatic acinar cell membranes and in electropermeabilized mouse pancreatic acini. J Pharmacol Exp Ther. 1990;253:847-850. 25. Saluja A, Saluja M, Printz H, Zavertnik A, Sengupta A, Steer ML. Experimental pancreatitis is mediated by low affinity CCK receptors which inhibit digestive enzyme secretion. Proc Natl Acad Sci U S A.1989;86:8969\x=req-\ 8971. 26. Saluja M, Saluja A, Lerch MM, Steer M. A plasma factor which is expressed during supramaximal stimulation causes in-vitro subcellular redistribution of lysosomal enzymes in rat exocrine pancreas. J Clin Invest. 1991; 87:1280-1285. 27. Lerch MM, Saluja A, Ramarao P, et al. Common pancreatic-bile duct (P-BD) obstruction in the opossum causes the early development of necrotizing pancreatitis. Pancreas. 1990;5:719.
Downloaded From: http://archsurg.jamanetwork.com/ by a University of Pittsburgh User on 06/03/2015
