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Early events in acute pancreatitis
BEST
Baillière’s Clinical Gastroenterology Vol. 13, No. 2, pp 213–225, 1999
B A I L L I È R E ’ S
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PRACTICE & RESEARCH
Early events in acute pancreatitis Michael L. Steer
MD
Professor of Surgery Harvard Medical School, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA
Studies using experimental models indicate that the earliest changes in acute pancreatitis involve intra-acinar cell events including the co-localization of lysosomal hydrolases with digestive enzyme zymogens. This co-localization phenomenon leads to trypsinogen activation and subsequent cell injury. Following acinar cell injury, a series of extra-acinar cell changes determine the severity of pancreatitis by promoting or reducing the inflammatory response and by influencing cell death events. Most patients with pancreatitis are diagnosed when acinar cell injury has already occurred. Therapies designed to modify the subsequent extra-acinar cell inflammatory process may prove useful in the management of clinical pancreatitis. Key words: experimental models; lysosomes; cathepsin B; digestive enzymes; trypsinogen; cytokines; chemokines; substance P; platelet activating factor; apoptosis.
Acute pancreatitis can be viewed as a disease which evolves in three phases—an initiating phase, a phase which involves acinar cell events including cell injury, and an extra-acinar cell phase in which the response to acinar cell injury includes pancreatic inflammation as well as systemic complications such as pulmonary and renal failure. The initiating phase of pancreatitis involves one or more processes which are often referred to as the ‘aetiologies’ of acute pancreatitis. These include biliary tract stone disease, ethanol abuse, pancreatic trauma or ischaemia, exposure to certain pancreaticotoxic drugs, hypercalcaemia, hypertriglyceridaemia, and the presence of lesions obstructing the pancreatic duct. The most common of these aetiologies, biliary tract stone disease, is believed to lead to pancreatitis by causing pancreatic duct outflow obstruction. This, combined with the observation that several otherwise dissimilar experimental models of acute pancreatitis are each characterized by defective pancreatic exocrine secretion, has suggested that such a phenomenon—i.e. an event which interferes with digestive enzyme secretion from pancreatic acinar cells—may be an early and critical event in the pathogenesis of all forms of acute pancreatitis. The second phase of acute pancreatitis appears to involve a series of changes which occur within pancreatic acinar cells. These changes culminate in the intra-acinar cell activation of digestive enzyme zymogens and acinar cell injury. Over the past two decades, our laboratory has undertaken a number of studies designed to characterize this phase of pancreatitis, and a description of our observations will form the major portion of this chapter. The third phase of pancreatitis involves a number of extra-acinar cell events, both within the pancreas and elsewhere in the body. To a great extent, these extra-acinar cell events are dependent on the local as well as systemic generation of inflammatory 1521–6918/99/020213 + 13 $12.00/00
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mediators, including cytokines, chemokines and other factors. These mediators regulate and co-ordinate the local inflammatory reaction, govern the type as well as the extent of pancreatic parenchymal cell death, and couple the local pancreatic injury with systemic complications such as pancreatitis-associated lung and/or renal injury. For the most part, patients with pancreatitis are identified hours, or even days, after the onset of their disease. Thus, by the time of diagnosis, the second phase of pancreatitis—i.e. intra-acinar cell digestive enzyme activation and acinar cell injury— has already been established and may no longer be responsive to therapeutic intervention. On the other hand, therapeutic interventions designed to alter or abort the extra-acinar cell events might still beneficially alter the course of clinical pancreatitis by reducing the extent of pancreatic inflammation and/or by minimizing the severity of extra-pancreatic complications. It is not surprising, therefore, that there have been a large number of recent studies designed to characterize this third phase of acute pancreatitis. Some of those studies, particularly those performed by our group, will be discussed in this chapter. EXPERIMENTAL MODELS OF ACUTE PANCREATITIS Studies designed to define the early events in acute pancreatitis cannot be performed using clinical material for several reasons, including (a) the highly complex and variable nature of clinical pancreatitis; (b) the relative inaccessibility of the human pancreas to experimental sampling; and (c) the fact that, for the most part, the early events in pancreatitis have already become established or are already completed prior to the time of clinical diagnosis. To overcome these problems, students of the disease have been forced to employ one or more models of acute pancreatitis established in experimental animals. Four types of model have been used: (a) the duct injection models in which bile, bile salts, and/or activated digestive enzymes are retrogradely injected into the pancreatic duct; (b) the duct obstruction models in which outflow of pancreatic exocrine secretions is mechanically prevented (Senninger et al, 1984); (c) the secretagogue-induced models in which rodents are given a dose of the cholecystokinin analogue caerulein that exceeds the dose which causes a maximal rate of digestive enzyme secretion (Lampel and Kern, 1977); and (d) the diet-induced model in which young female mice are given a choline-deficient diet that is supplemented with the ethyl analogue of methionine, ethionine (Lombardi et al, 1975). A defect in digestive enzyme secretion from the pancreas is a common feature in most, if not all, of these models. In the duct obstruction models, drainage of pancreatic secretions into the intestinal tract is prevented by a surgically placed ligature (Lerch et al, 1995). In the secretagogue-induced models, the supramaximally stimulating concentrations of caerulein which are administered interact with low-affinity CCK-Areceptors on acinar cells and, by as yet undefined mechanisms, this interaction results in the inhibition of secretion (Saluja et al, 1989). In the diet-induced model, administration of the choline-deficient ethionine-supplemented (CDE) diet induces a defect in acinar cell stimulus-secretion coupling, and, as a result, digestive enzymes can be synthesized by acinar cells but not secreted in response to secretagogue stimulation (Powers et al, 1986). The severity of pancreatitis induced in each of these types of model varies considerably. Retrograde ductal injection results in rapid, massive and widespread pancreatic injury with an associated high mortality rate. Duct obstruction in most experimental animals causes exocrine pancreatic atrophy with only mild inflammation
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and no death, but in the American opossum, pancreatic duct obstruction causes a highly lethal, severe, necrotizing and haemorrhagic pancreatitis. In that species (and perhaps in humans), the disease progresses over several days and its severity is directly related to the duration of duct obstruction (Lerch et al, 1993b). Secretagogue-induced pancreatitis in rodents evolves within several hours of the onset of supramaximal stimulation and it is characterized by hyperamylasaemia, pancreatic oedema, acinar cell vacuolization and variable degrees of pancreatic inflammation but little or no mortality (Grady et al, 1996). Finally, diet-induced pancreatitis in young female mice is characterized by marked necrosis and haemorrhage that evolve over several days, and a mortality rate which is dependent upon the amount as well as the duration of CDE diet administration (Lombardi et al, 1975).
ACINAR CELL BIOLOGY The earliest events in the evolution of acute pancreatitis involve perturbations of normal acinar cell biology. Thus, an understanding of those events is dependent upon our knowledge of normal acinar cell biology. The pancreatic acinar cell is a virtual factory for protein synthesis. Those proteins are assembled in the rough endoplasmic reticulum (RER), and vectorially transported to the Golgi complex where post-translational modifications occur. More than 90% of the protein synthesized by acinar cells consists of digestive enzymes which are destined to be transported out of the cell. Those proteins, which exist primarily as inactive pro-enzymes or zymogens, are packaged in condensing vacuoles at the trans side of the Golgi and carried towards the lumenal plasma membrane. The condensing vacuoles evolve into zymogen granules as the electron density of their contents increases. At the plasma membrane, those zymogen granules fuse with the surface membrane and, by fusion–fission, release their contents into the acinar (i.e. ductal) space (Palade, 1975). Acinar cells also synthesize enzymes which are destined to be transported, intracellularly, to lysosomes where they act to digest intracellular substrates. As they traverse the Golgi complex, these lysosomal hydrolases are phosphorylated at the 6-position of mannose residues, segregated from secretory proteins by being bound to receptors in the Golgi complex which recognize the mannose-6-phosphate label, and then transported away from the secretory pathway to the lysosomal compartment (Kornfield, 1986).
ACINAR CELL PATHOBIOLOGY IN ACUTE PANCREATITIS Our studies (see Steer et al, 1998 for a recent review), designed to explore the cell biological changes that occur during the early stages of pancreatitis, have been performed using the duct obstruction model of pancreatitis in opossums, the dietinduced model in mice, and the secretagogue-induced models in both rats and mice. In each of these models, we have found that digestive enzyme synthesis is unaltered but digestive enzyme secretion from acinar cells is blocked (Figure 1). We have also found that digestive enzyme segregation from lysosomal hydrolases during intracellular transport is disturbed and, as a result, digestive zymogens became co-localized with lysosomal hydrolases in intracellular vacuoles. This co-localization phenomenon has been documented by immunolocalization studies at both the light microscope
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Figure 1. Protein synthesis and discharge. Rats were pre-infused with saline alone (filled circles) or saline containing sufficient caerulein to deliver 5 µg/kg/hour (open circles) for 1 hour, given a pulse of [3H]phenylalanine followed by a bolus of non-radioactive phenylalanine, and continued on infusion for varying times. At selected intervals, rats in each group were killed, and trichloroacetic acid-precipitable radioactivity was measured in pancreas homogenates. Asterisks denote values in caerulein-infused animals that are significantly different from those found in saline-infused rats. Reprinted from Saluja et al (1985, American Journal of Physiology 249: G702–G710) with permission.
(Figure 2) and electron microscope level of resolution. It has also been shown in subcellular fractionation studies in which the lysosomal hydrolases were found to move from the lysosomal fraction into a heavier fraction that is enriched in zymogen granules (Figure 3). These observations suggested that the co-localization of lysosomal hydrolases with digestive enzyme zymogens might be an early and critical event in the evolution of pancreatitis. Indeed, observations by others (Greenbaum and Hirschkowitz, 1961; Figarella et al, 1988) which were confirmed by us (Lerch et al, 1993a) indicated that, under appropriate conditions, the lysosomal hydrolase cathepsin B can activate trypsinogen, and active trypsin is known to be capable of activating the other zymogens. We suspected that intra-acinar cell activation of trypsinogen might lead to cell injury if the activated protease were released into the cytoplasmic space (Figure 4). Although the co-localization of digestive enzyme zymogens with lysosomal hydrolases during the early stages of experimental pancreatitis has been repeatedly observed by us as well as by others, the importance of this phenomenon to the evolution of pancreatitis has not been uniformly accepted. In order to establish a causal relationship between the co-localization phenomenon and pancreatitis, we concluded that the following four issues must be resolved: (a) the co-localization phenomenon must precede evidence of cell injury; (b) digestive enzyme zymogen activation must occur at the site of co-localization; (c) inhibition of lysosomal hydrolases such as cathepsin B should prevent zymogen activation; and (d) prevention of zymogen activation and/or inhibition of the activated zymogens should prevent cell injury. Our recently completed studies (see below) indicate that each of these four relationships exist.
Figure 2. Indirect immunofluorescence of pancreatic acinar cell of rats infused with caerulein for 1 hour. The two panels illustrate immunolabelling obtained with antizymogen (A) and anticathepsin B (B) serum, respectively, in corresponding fields from two semithin (1-µm) sections cut next to each other from the same block. Antizymogen immunolabelling is diffuse throughout the cytoplasm, with heavy reaction of zymogen granules (localized at the cell apex) and, especially, on large, heterogeneous granules localized preferentially in the Golgi area. Anticathepsin labelling is restricted to vacuoles with the latter localization. Numbers (1–10) point to vacuoles in two adjacent sections that were labelled by both sera used. Original magnification, ×1200). Reprinted from Watanabe et al (1984, American Journal of Physiology 246: G457–G467) with permission.
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Figure 3. Subcellular distribution of cathepsin B. Rats were infused with saline (open bars) or saline containing caerulein at a concentration designed to deliver either 0.25 µg/kg/hour (filled bars) or 5 µg/kg/hour (hatched bars) for 3.5 hours prior to death, and the pancreas was subcellularly fractionated by differential centrifugation. Cathepsin B activity, measured in each fraction, is expressed as a percentage of the total found in the post-nuclear homogenate. 1.3KP, 1300 g × 15 minutes pellet (primarily zymogen granules); 12KP, 12 000 g × 12 minutes pellet (primarily lysosomes and mitochondria); 105KP, 105 000 g × 60 minutes pellet (primarily microsomes); 105KS, 105 000 g × 60 minutes supernatant (soluble fraction). Results represent mean values and vertical bars represent standard errors obtained from four or more separate fractionations, each performed using samples from different animals. Reprinted from Saluja et al (1987) with permission.
Timing of events The secretagogue-induced model of pancreatitis was used to determine whether the co-localization phenomenon and trypsinogen activation in the pancreas precedes other changes of pancreatitis. As can be seen in Table 1, subcellular redistribution of cathepsin B from the lysosomal to the zymogen granule compartment, release of trypsinogen activation peptide (indicative of trypsinogen activation), and the appearance of active trypsin in the pancreas can be detected within 15 minutes of the start of supramaximal secretagogue stimulation, while hyperamylasemia, pancreatic oedema and depletion of pancreatic glutathione levels occur at later times (Grady et al, 1996). Thus, as predicted by the co-localization hypothesis, the co-localization phenomenon and trypsinogen activation appear to precede other early changes of pancreatitis in this model.
Table 1. Timing of events noted after onset of supramaximal caerulein infusion in rats. Event
Time
Enzyme redistribution Activated trypsinogen Free trypsinogen activation peptide Hyperamylasaemia Pancreatic oedema Acinar cell vacuolization Glutathione depletion
15 10 30 30 60 60 >60
Transport
Digestive enzymes in condensing vacuoles
Lysosomal and digestive enzymes co-localized
No segregation
To Golgi complex
Segregation
Digestive enzyme activation by lysosomal enzymes in fragile vacuoles
Zymogen granules
Release of active enzymes in cytoplasm
Rupture of vacuoles
at plasmalemma
Fusion/fission
Acinar cell injury
Digestive enzyme secretion into pancreatic duct
Figure 4. Theoretical scheme for cellular events leading to pancreatitis. The co-localization hypothesis. Reprinted from Steer (1995) with permission.
PANCREATITIS
Digestive and lysosomal enzyme synthesis in endoplasmic reticulum
NORMAL
Lysosomal enzymes in lysosomes
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Location of activation Time-dependent studies using the secretagogue-induced model were also performed to identify the site of intra-acinar cell trypsinogen activation (Hofbauer et al, 1998b). Using the technique of subcellular fractionation, active trypsin and the trypsinogen activation peptide were, at the earliest times, located within the subcellular fraction which contained co-localized lysosomal and digestive enzymes. Immunolocalization studies performed at the electron microscope level of resolution indicated that TAP and cathepsin B were both contained within the same cytoplasmic vacuoles at the earliest times after the onset of supramaximal secretagogue stimulation (Hofbauer et al, 1998b). Thus, as predicted by the co-localization hypothesis, trypsinogen activation appears to occur within the vacuoles which contain co-localized lysosomal hydrolases and digestive zymogens in this model. Role of cathepsin B Attempts to abort intra-pancreatic activation of trypsinogen and the development of pancreatitis by administration of cathepsin B inhibitors to intact animals were unsuccessful because complete inhibition of pancreatic cathepsin B cannot be achieved in vivo (Saluja et al, 1991). To overcome this problem, we established an in vitro model to replicate the events in secretagogue-induced pancreatitis. We found that intra-
Figure 5. Time course of caerulein-induced activation of trypsinogen. Rat pancreatic acini were incubated with either buffer alone (filled circles) or buffer containing 0.1 µM cerulein (filled squares) for varying times, and the trypsin activity in the homogenized acini was measured. Results are expressed as a percentage of the maximal activity obtained in each experiment. Values shown are expressed as means ± SEM obtained from at least four experiments. *P <0.05 compared with time 0. Reprinted from Saluja et al (1997) with permission.
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acinar cell activation of trypsinogen can be detected when freshly prepared but otherwise normal pancreatic acini are incubated for 15–30 minutes in vitro with a supramaximally stimulating concentration of caerulein (Figure 5) and that activation can be prevented by inclusion of the cathepsin B inhibitor E-64D in the incubation mixture (Figure 6) (Saluja et al, 1997). Thus, as predicted by the co-localization hypothesis, complete inhibition of cathepsin B can prevent trypsinogen activation.
120
Enzyme activity (% of maximal)
100
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60
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20 (0)
0 BASAL
CER
CER + 1mM E-64d
Figure 6. Effect of E-64d on cathepsin B activity and caerulein (CER)-induced activation of trypsinogen. After a 30-minute pre-incubation with 1 mM cathepsin B inhibitor E-64d, rat pancreatic acini were further incubated with 0.1 µM CER together with E-64d for 30 minutes, and cathepsin B (hatched bars) and trypsin (filled bars) activities in the homogenized acini were measured. Results are expressed as a percentage of the activity obtained after incubation with CER alone. Values shown are means ± SEM obtained from at least three experiments. Reprinted from Saluja et al (1997, Gastroenterology 113: 304–310) with permission.
Cell injury The in vitro system that involves incubating freshly prepared rat pancreatic acini with a supramaximally stimulating concentration of caerulein was also used to evaluate the relationship between intra-acinar cell activation of trypsinogen and acinar cell injury. Trypsinogen activation was found to be dependent upon a secretagogue-induced rise in intracellular ionized calcium and to precede evidence of cell injury. Prevention of secretagogue-induced trypsinogen activation or, alternatively, inhibition of trypsin activity, was found to prevent acinar cell injury (Saluja et al, unpublished observations). These observations, therefore, support the co-localization hypothesis by demonstrating that the prevention of cathepsin B-mediated trypsinogen activation and/or inhibition of active trypsin within acinar cells can prevent secretagogue-induced cell injury.
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DETERMINANTS OF SEVERITY The studies summarized above support the hypothesis that the earliest changes in pancreatitis occur within acinar cell cytoplasmic vacuoles where the co-localization of digestive enzyme zymogens with lysosomal hydrolases leads to trypsinogen activation, activation of the other zymogens, and ultimately to acinar cell injury. The colocalization phenomenon and zymogen activation are common features shared by many of the models of pancreatitis, yet the severity of these models varies considerably. Similarly, clinical pancreatitis can be either mild or severe. This had suggested that events which occur subsequent to intra-acinar cell activation of zymogens might play an important role in regulating the severity of pancreatitis. Apoptosis/necrosis Cell death may occur as a result of either apoptosis or necrosis. The former is an energy dependent, genetically programmed process which usually involves little or no inflammation. In contrast, necrosis is usually accompanied by an extensive inflammatory reaction. The events which underlie pancreatic acinar cell apoptosis have not been extensively characterized but studies to date indicate that it is, at least in part, triggered by tumour necrosis factor-α, generated by neutrophils and, perhaps, by acinar cells themselves (Gukovskaya et al, 1997). As in other types of cell, apoptosis in acinar cells involves the internucleosomal degradation of genomic DNA catalysed by a Ca2+-Mg2+-dependent endonuclease. We (Kaiser et al, 1995) and others (Gukovskaya et al, 1997) have found that extensive apoptosis of acinar cells occurs in models of mild pancreatitis, whereas acinar cell necrosis is a characteristic of severe pancreatitis. Furthermore, interventions that promote apoptosis lead to a reduction in the severity of pancreatitis (Saluja et al, 1996; Bhatia et al, 1998b) while those that prevent apoptosis cause a worsening of pancreatitis (Kaiser et al, 1996). This has suggested that apoptosis might be a teleologically favourable response to acinar cell injury in pancreatitis and that therapeutic interventions designed to promote acinar cell apoptosis might prove to be beneficial in the management of patients with clinical pancreatitis. Mediators of inflammation The local as well as the systemic changes of pancreatitis are closely regulated by a vast array of factors which co-ordinate and control the inflammatory response. Considerable recent attention has been directed at identifying and characterizing those factors, but our knowledge concerning this complex process remains very limited. Until recently most studies in this area were performed using inhibitors designed to block the effects of putative pro-inflammatory factors. More recently, targeted gene deletion has been employed to generate knock-out mice which lack receptors specific for those factors or which cannot generate the factors themselves. Using these, as well as other methods, Norman and co-workers (Denham et al, 1997) as well as Pandol and his colleagues (Gukovskaya et al, 1997) have shown that inteleukin-1 (IL-1) and TNF-α play important pro-inflammatory roles in pancreatitis. Reber and his group (Rongione et al, 1997) as well as van Laethem et al (1995) have shown that IL-10 exerts a potent anti-inflammatory influence in pancreatitis. We (Hofbauer et al, in press) and others (Formela et al, 1994) have found that platelet activating factor (PAF) has a proinflammatory effect in experimental pancreatitis and that either inhibition of PAF
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action or hydrolysis of PAF can protect against pancreatitis and pancreatitis-associated lung injury. The neurokinin substance P, acting via neurokinin-1 receptors (NK-1r), which normally functions as a neuraltransmitter in afferent vagal fibres from the pancreas, has been found to exert a potent pro-inflammatory effect in pancreatitis, mediating so-called neurogenic inflammation in the pancreas and pancreatitisassociated lung injury (Bhatia et al, 1998a) (Figure 7). Chemokines also play important roles in regulating the local and systemic changes in pancreatitis by controlling the activation and recruitment of inflammatory cells. Mice which lack the C-C chemokine receptor CCR-1 are protected against pancreatitis-associated lung injury (Figure 8) (Gerard et al, 1997). Finally, complement and the factors generated during complement activation are believed to play important roles in pancreatitis. The nature of that role, however, is far from clear as shown by our recent observation that the anaphylatoxin C5a, which would be expected to play a pro-inflammatory role in pancreatitis, actually has a dramatic anti-inflammatory effect (Bhatia et al, unpublished observations).
Figure 7. Effects of NK1R deletion on pancreatitis-associated lung injury. Mice were given 12 hourly injections of caerulein (50 µg/kg intraperitoneally). One hour after the last injection of caerulein, mice were killed and the capillary leakage of FITC-albumin and lung MPO activity were measured. Values are expressed as a percentage of the value obtained for wild-type animals given caerulein. These values (100%) were as follows: lung MPO activity: 3.06 ± 0.37; FITC-albumin bronchoalveolar lavage fluid to serum ratio 2.13 ± 0.24. Results shown are the mean ± SEM for 10 or more animals in each group. Asterisks indicate P < 0.05 when NK1R–/– animals were compared with NK1R+/+ animals. Reprinted from Bhatia et al (1998, Proceedings of the National Academy of Sciences of the USA 95: 4760–4765) with permission.
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Figure 8. Effects of CCR1 receptor deletion on pancreatitis-associated lung injury. Capillary leakage of FITC-albumin and lung MPO activity were measured as described in the text. Solid bars indicate wild-type control animals, open bars indicate CCR1 receptor deficient control animals, single-hatched bars indicate wild-type animals given 12 hourly injections of a supramaximally stimulating dose of caerulein, and doublehatched bars indicate CCR1 receptor-deficient animals given 12 hourly injections of caerulein. Values are expressed as a percentage of the value obtained for wild-type animals given caerulein. Results shown are mean ± SEM values for 10 or more animals in each group. *P <0.05 when CCR1 receptor-deficient animals were compared with wild-type animals. Reprinted from Gerard et al (1997, Journal of Clinical Investigation 100: 2022–2027) with permission.
CONCLUSION Acute pancreatitis is a complex disease which can be initiated by a variety of events. The earliest changes in pancreatitis occur within acinar cells where activation of digestive enzyme zymogens leads to acinar cell injury. Subsequent events, occurring as a result of acinar cell injury, regulate the severity of pancreatitis and the systemic response, including the associated lung injury. Future studies are likely to broaden our understanding of these various processes and, in this way, suggest therapies which might either abort or reduce the severity of clinical pancreatitis. REFERENCES *Bhatia M, Saluja AK, Hofbauer B et al (1998a) Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury. Proceedings of the National Academy of Sciences of the USA 95: 4760–4765. Bhatia M, Wallig M, Hofbauer B et al (1998b) Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis. Biochemical and Biophysical Research Communications 246: 476–483. Denham W, Yang J, Fink G et al (1997) Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis. Gastroenterology 113: 1741–1746. Figarella C, Miszczuk-Jamska & Barrett AL (1998) Possible lusosomal activation of pancreatic zymogens: activation of both human trypsinogen by cathepsin B and spontaneous acid activation of human trypsinogen 1. Hoppe Seyler’s Journal of Biological Chemistry 369: 293–298.
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Formela LJ, Wood LM, Whittaker M et al (1994) Amelioration of experimental acute pancreatitis with a potent platelet-activating factor antagonist. British Journal of Surgery 81: 1783–1785. *Gerard C, Frossard JL, Bhatia M et al (1997) Targeted disruption of the B-chemokine receptor CCRI protects against pancreatitis-associated lung injury. Journal of Clinical Investigation 100: 2022–2027. *Grady T, Saluja A, Kaiser A et al (1996) Pancreatic edema and intrapancreatic activation of trypsinogen during secretagogue-induced pancreatitis precedes glutathione depletion. American Journal of Physiology 271: G20–G26. Greenbaum LA & Hirschkowitz A (1961) Endogenous cathepsin activated trypsinogen in extracts of dog pancreas. Proceedings of the Society of Experimental Biology and Medicine 107: 74–76. *Gukovskaya AS, Gukovsky I, Zaninovic V et al (1997) Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis. Journal of Clinical Investigation 100: 1853–1862. Hofbauer B, Saluja A, Bhatia M et al (1998a) Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis. Gastroenterology 115: 1238–1247. *Hofbauer B, Saluja AK, Lerch M et al (1998b) Intra-acinar cell activation of trypsinogen during caeruleininduced pancreatitis in rats. American Journal of Physiology 275: G352–G362. Kaiser A, Saluja A, Sengupta A et al (1995) Relationship between severity, necrosis and apoptosis in five models of experimental acute pancreatitis. American Journal of Physiology 38: C1295–C1304. Kaiser AM, Saluja AK, Lu L et al (1996) Effects of cyclohexamide on pancreatic endonuclease activity, acinar cell apoptosis, and the severity of acute pancreatitis. American Journal of Physiology 40: C982. Kornfield S (1986) Trafficking of lysosomal enzymes in normal and disease states. Journal of Clinical Investigation 77: 1–6. van Laethem JL, Marchant A, Delvaux A et al (1995) Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 108: 1917–1922. *Lampel M & Kern H (1977) Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Archiv 373: 97–117. Lerch MM, Saluja AK, Dawra R et al (1993a) The effect of choloroquine administration on two experimental models of acute pancreatitis. Gastroenterology 104: 1768–1779. *Lerch MM, Saluja A, Runzi M et al (1993b) Pancreatitis duct obstruction triggers acute necrotizing pancreatitis in the opossum. Gastroenterology 104: 853–861. Lerch M, Saluja A, Kaiser A et al (1995) Luminal endocytosis and intracellular targeting of acinar cells during early biliary pancreatitis in the opossum. Journal of Clinical Investigation 95: 2222–2231. Lombardi B, Estes LW & Longnecker DW (1975) Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet. American Journal of Pathology 79: 465–480. Palade G (1975) Intracellular aspects of the process of protein secretion. Science 189: 347–358. Powers RE, Saluja AK, Houlihan ML et al (1986) Diminished agonist-stimulated inositrol trisphosphate generation blocks stimulatus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreatitis. Journal of Clinical Investigation 77: 1668–1674. Rongione AJ, Kusske AM, Kwan K et al (1997) Interleukin 10 reduces the severity of acute pancreatitis in rats. Gastroenterology 112: 960–967. Saluja A, Hashimoto S, Saluja M et al (1987) Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. American Journal of Physiology 253: G508–G516. *Saluja A, Saluja M, Printz H et al (1989) Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion. Proceedings of the National Academy of Sciences of the USA 86: 347–358. Saluja A, Steer D, Lerch MM et al (1991) Failure of the cathepsin B inhibitor, E-64, to protect against caerulein and CDE-diet induced pancreatitis. Pancreas 6: 718. Saluja A, Hofbauer B, Yamaguchi Y et al (1996) Induction of apoptosis reduces the severity of caeruleininduced pancreatitis in mice. Biochemical and Biophysical Research Communications 220: 875–878. *Saluja AK, Donovan EA, Yamanaka K et al (1997) Caerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B. Gastroenterology 113: 304–310. Senninger N, Moody FG, van Buren DH et al (1984) Effect of biliary obstruction on pancreatic exocrine secretion in conscious opossums. Surgical Forum 35: 226–228. Steer ML (1995) Recent insights into the etiology and pathogenesis of acute biliary pancreatitis. American Journal of Roentgenology 164: 811–814. *Steer ML (1998) The early intra-acinar cell events which occur during acute pancreatitis. Pancreas 17: 31–37. Watanabe O, Baccino FM, Steer ML & Meldolesi J (1984) Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis. American Journal of Physiology 246: G457–G467.
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Baillière’s Clinical Gastroenterology Vol. 13, No. 2, pp 213–225, 1999
B A I L L I È R E ’ S
1
PRACTICE & RESEARCH
Early events in acute pancreatitis Michael L. Steer
MD
Professor of Surgery Harvard Medical School, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA
Studies using experimental models indicate that the earliest changes in acute pancreatitis involve intra-acinar cell events including the co-localization of lysosomal hydrolases with digestive enzyme zymogens. This co-localization phenomenon leads to trypsinogen activation and subsequent cell injury. Following acinar cell injury, a series of extra-acinar cell changes determine the severity of pancreatitis by promoting or reducing the inflammatory response and by influencing cell death events. Most patients with pancreatitis are diagnosed when acinar cell injury has already occurred. Therapies designed to modify the subsequent extra-acinar cell inflammatory process may prove useful in the management of clinical pancreatitis. Key words: experimental models; lysosomes; cathepsin B; digestive enzymes; trypsinogen; cytokines; chemokines; substance P; platelet activating factor; apoptosis.
Acute pancreatitis can be viewed as a disease which evolves in three phases—an initiating phase, a phase which involves acinar cell events including cell injury, and an extra-acinar cell phase in which the response to acinar cell injury includes pancreatic inflammation as well as systemic complications such as pulmonary and renal failure. The initiating phase of pancreatitis involves one or more processes which are often referred to as the ‘aetiologies’ of acute pancreatitis. These include biliary tract stone disease, ethanol abuse, pancreatic trauma or ischaemia, exposure to certain pancreaticotoxic drugs, hypercalcaemia, hypertriglyceridaemia, and the presence of lesions obstructing the pancreatic duct. The most common of these aetiologies, biliary tract stone disease, is believed to lead to pancreatitis by causing pancreatic duct outflow obstruction. This, combined with the observation that several otherwise dissimilar experimental models of acute pancreatitis are each characterized by defective pancreatic exocrine secretion, has suggested that such a phenomenon—i.e. an event which interferes with digestive enzyme secretion from pancreatic acinar cells—may be an early and critical event in the pathogenesis of all forms of acute pancreatitis. The second phase of acute pancreatitis appears to involve a series of changes which occur within pancreatic acinar cells. These changes culminate in the intra-acinar cell activation of digestive enzyme zymogens and acinar cell injury. Over the past two decades, our laboratory has undertaken a number of studies designed to characterize this phase of pancreatitis, and a description of our observations will form the major portion of this chapter. The third phase of pancreatitis involves a number of extra-acinar cell events, both within the pancreas and elsewhere in the body. To a great extent, these extra-acinar cell events are dependent on the local as well as systemic generation of inflammatory 1521–6918/99/020213 + 13 $12.00/00
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mediators, including cytokines, chemokines and other factors. These mediators regulate and co-ordinate the local inflammatory reaction, govern the type as well as the extent of pancreatic parenchymal cell death, and couple the local pancreatic injury with systemic complications such as pancreatitis-associated lung and/or renal injury. For the most part, patients with pancreatitis are identified hours, or even days, after the onset of their disease. Thus, by the time of diagnosis, the second phase of pancreatitis—i.e. intra-acinar cell digestive enzyme activation and acinar cell injury— has already been established and may no longer be responsive to therapeutic intervention. On the other hand, therapeutic interventions designed to alter or abort the extra-acinar cell events might still beneficially alter the course of clinical pancreatitis by reducing the extent of pancreatic inflammation and/or by minimizing the severity of extra-pancreatic complications. It is not surprising, therefore, that there have been a large number of recent studies designed to characterize this third phase of acute pancreatitis. Some of those studies, particularly those performed by our group, will be discussed in this chapter. EXPERIMENTAL MODELS OF ACUTE PANCREATITIS Studies designed to define the early events in acute pancreatitis cannot be performed using clinical material for several reasons, including (a) the highly complex and variable nature of clinical pancreatitis; (b) the relative inaccessibility of the human pancreas to experimental sampling; and (c) the fact that, for the most part, the early events in pancreatitis have already become established or are already completed prior to the time of clinical diagnosis. To overcome these problems, students of the disease have been forced to employ one or more models of acute pancreatitis established in experimental animals. Four types of model have been used: (a) the duct injection models in which bile, bile salts, and/or activated digestive enzymes are retrogradely injected into the pancreatic duct; (b) the duct obstruction models in which outflow of pancreatic exocrine secretions is mechanically prevented (Senninger et al, 1984); (c) the secretagogue-induced models in which rodents are given a dose of the cholecystokinin analogue caerulein that exceeds the dose which causes a maximal rate of digestive enzyme secretion (Lampel and Kern, 1977); and (d) the diet-induced model in which young female mice are given a choline-deficient diet that is supplemented with the ethyl analogue of methionine, ethionine (Lombardi et al, 1975). A defect in digestive enzyme secretion from the pancreas is a common feature in most, if not all, of these models. In the duct obstruction models, drainage of pancreatic secretions into the intestinal tract is prevented by a surgically placed ligature (Lerch et al, 1995). In the secretagogue-induced models, the supramaximally stimulating concentrations of caerulein which are administered interact with low-affinity CCK-Areceptors on acinar cells and, by as yet undefined mechanisms, this interaction results in the inhibition of secretion (Saluja et al, 1989). In the diet-induced model, administration of the choline-deficient ethionine-supplemented (CDE) diet induces a defect in acinar cell stimulus-secretion coupling, and, as a result, digestive enzymes can be synthesized by acinar cells but not secreted in response to secretagogue stimulation (Powers et al, 1986). The severity of pancreatitis induced in each of these types of model varies considerably. Retrograde ductal injection results in rapid, massive and widespread pancreatic injury with an associated high mortality rate. Duct obstruction in most experimental animals causes exocrine pancreatic atrophy with only mild inflammation
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and no death, but in the American opossum, pancreatic duct obstruction causes a highly lethal, severe, necrotizing and haemorrhagic pancreatitis. In that species (and perhaps in humans), the disease progresses over several days and its severity is directly related to the duration of duct obstruction (Lerch et al, 1993b). Secretagogue-induced pancreatitis in rodents evolves within several hours of the onset of supramaximal stimulation and it is characterized by hyperamylasaemia, pancreatic oedema, acinar cell vacuolization and variable degrees of pancreatic inflammation but little or no mortality (Grady et al, 1996). Finally, diet-induced pancreatitis in young female mice is characterized by marked necrosis and haemorrhage that evolve over several days, and a mortality rate which is dependent upon the amount as well as the duration of CDE diet administration (Lombardi et al, 1975).
ACINAR CELL BIOLOGY The earliest events in the evolution of acute pancreatitis involve perturbations of normal acinar cell biology. Thus, an understanding of those events is dependent upon our knowledge of normal acinar cell biology. The pancreatic acinar cell is a virtual factory for protein synthesis. Those proteins are assembled in the rough endoplasmic reticulum (RER), and vectorially transported to the Golgi complex where post-translational modifications occur. More than 90% of the protein synthesized by acinar cells consists of digestive enzymes which are destined to be transported out of the cell. Those proteins, which exist primarily as inactive pro-enzymes or zymogens, are packaged in condensing vacuoles at the trans side of the Golgi and carried towards the lumenal plasma membrane. The condensing vacuoles evolve into zymogen granules as the electron density of their contents increases. At the plasma membrane, those zymogen granules fuse with the surface membrane and, by fusion–fission, release their contents into the acinar (i.e. ductal) space (Palade, 1975). Acinar cells also synthesize enzymes which are destined to be transported, intracellularly, to lysosomes where they act to digest intracellular substrates. As they traverse the Golgi complex, these lysosomal hydrolases are phosphorylated at the 6-position of mannose residues, segregated from secretory proteins by being bound to receptors in the Golgi complex which recognize the mannose-6-phosphate label, and then transported away from the secretory pathway to the lysosomal compartment (Kornfield, 1986).
ACINAR CELL PATHOBIOLOGY IN ACUTE PANCREATITIS Our studies (see Steer et al, 1998 for a recent review), designed to explore the cell biological changes that occur during the early stages of pancreatitis, have been performed using the duct obstruction model of pancreatitis in opossums, the dietinduced model in mice, and the secretagogue-induced models in both rats and mice. In each of these models, we have found that digestive enzyme synthesis is unaltered but digestive enzyme secretion from acinar cells is blocked (Figure 1). We have also found that digestive enzyme segregation from lysosomal hydrolases during intracellular transport is disturbed and, as a result, digestive zymogens became co-localized with lysosomal hydrolases in intracellular vacuoles. This co-localization phenomenon has been documented by immunolocalization studies at both the light microscope
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Figure 1. Protein synthesis and discharge. Rats were pre-infused with saline alone (filled circles) or saline containing sufficient caerulein to deliver 5 µg/kg/hour (open circles) for 1 hour, given a pulse of [3H]phenylalanine followed by a bolus of non-radioactive phenylalanine, and continued on infusion for varying times. At selected intervals, rats in each group were killed, and trichloroacetic acid-precipitable radioactivity was measured in pancreas homogenates. Asterisks denote values in caerulein-infused animals that are significantly different from those found in saline-infused rats. Reprinted from Saluja et al (1985, American Journal of Physiology 249: G702–G710) with permission.
(Figure 2) and electron microscope level of resolution. It has also been shown in subcellular fractionation studies in which the lysosomal hydrolases were found to move from the lysosomal fraction into a heavier fraction that is enriched in zymogen granules (Figure 3). These observations suggested that the co-localization of lysosomal hydrolases with digestive enzyme zymogens might be an early and critical event in the evolution of pancreatitis. Indeed, observations by others (Greenbaum and Hirschkowitz, 1961; Figarella et al, 1988) which were confirmed by us (Lerch et al, 1993a) indicated that, under appropriate conditions, the lysosomal hydrolase cathepsin B can activate trypsinogen, and active trypsin is known to be capable of activating the other zymogens. We suspected that intra-acinar cell activation of trypsinogen might lead to cell injury if the activated protease were released into the cytoplasmic space (Figure 4). Although the co-localization of digestive enzyme zymogens with lysosomal hydrolases during the early stages of experimental pancreatitis has been repeatedly observed by us as well as by others, the importance of this phenomenon to the evolution of pancreatitis has not been uniformly accepted. In order to establish a causal relationship between the co-localization phenomenon and pancreatitis, we concluded that the following four issues must be resolved: (a) the co-localization phenomenon must precede evidence of cell injury; (b) digestive enzyme zymogen activation must occur at the site of co-localization; (c) inhibition of lysosomal hydrolases such as cathepsin B should prevent zymogen activation; and (d) prevention of zymogen activation and/or inhibition of the activated zymogens should prevent cell injury. Our recently completed studies (see below) indicate that each of these four relationships exist.
Figure 2. Indirect immunofluorescence of pancreatic acinar cell of rats infused with caerulein for 1 hour. The two panels illustrate immunolabelling obtained with antizymogen (A) and anticathepsin B (B) serum, respectively, in corresponding fields from two semithin (1-µm) sections cut next to each other from the same block. Antizymogen immunolabelling is diffuse throughout the cytoplasm, with heavy reaction of zymogen granules (localized at the cell apex) and, especially, on large, heterogeneous granules localized preferentially in the Golgi area. Anticathepsin labelling is restricted to vacuoles with the latter localization. Numbers (1–10) point to vacuoles in two adjacent sections that were labelled by both sera used. Original magnification, ×1200). Reprinted from Watanabe et al (1984, American Journal of Physiology 246: G457–G467) with permission.
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Figure 3. Subcellular distribution of cathepsin B. Rats were infused with saline (open bars) or saline containing caerulein at a concentration designed to deliver either 0.25 µg/kg/hour (filled bars) or 5 µg/kg/hour (hatched bars) for 3.5 hours prior to death, and the pancreas was subcellularly fractionated by differential centrifugation. Cathepsin B activity, measured in each fraction, is expressed as a percentage of the total found in the post-nuclear homogenate. 1.3KP, 1300 g × 15 minutes pellet (primarily zymogen granules); 12KP, 12 000 g × 12 minutes pellet (primarily lysosomes and mitochondria); 105KP, 105 000 g × 60 minutes pellet (primarily microsomes); 105KS, 105 000 g × 60 minutes supernatant (soluble fraction). Results represent mean values and vertical bars represent standard errors obtained from four or more separate fractionations, each performed using samples from different animals. Reprinted from Saluja et al (1987) with permission.
Timing of events The secretagogue-induced model of pancreatitis was used to determine whether the co-localization phenomenon and trypsinogen activation in the pancreas precedes other changes of pancreatitis. As can be seen in Table 1, subcellular redistribution of cathepsin B from the lysosomal to the zymogen granule compartment, release of trypsinogen activation peptide (indicative of trypsinogen activation), and the appearance of active trypsin in the pancreas can be detected within 15 minutes of the start of supramaximal secretagogue stimulation, while hyperamylasemia, pancreatic oedema and depletion of pancreatic glutathione levels occur at later times (Grady et al, 1996). Thus, as predicted by the co-localization hypothesis, the co-localization phenomenon and trypsinogen activation appear to precede other early changes of pancreatitis in this model.
Table 1. Timing of events noted after onset of supramaximal caerulein infusion in rats. Event
Time
Enzyme redistribution Activated trypsinogen Free trypsinogen activation peptide Hyperamylasaemia Pancreatic oedema Acinar cell vacuolization Glutathione depletion
15 10 30 30 60 60 >60
Transport
Digestive enzymes in condensing vacuoles
Lysosomal and digestive enzymes co-localized
No segregation
To Golgi complex
Segregation
Digestive enzyme activation by lysosomal enzymes in fragile vacuoles
Zymogen granules
Release of active enzymes in cytoplasm
Rupture of vacuoles
at plasmalemma
Fusion/fission
Acinar cell injury
Digestive enzyme secretion into pancreatic duct
Figure 4. Theoretical scheme for cellular events leading to pancreatitis. The co-localization hypothesis. Reprinted from Steer (1995) with permission.
PANCREATITIS
Digestive and lysosomal enzyme synthesis in endoplasmic reticulum
NORMAL
Lysosomal enzymes in lysosomes
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Location of activation Time-dependent studies using the secretagogue-induced model were also performed to identify the site of intra-acinar cell trypsinogen activation (Hofbauer et al, 1998b). Using the technique of subcellular fractionation, active trypsin and the trypsinogen activation peptide were, at the earliest times, located within the subcellular fraction which contained co-localized lysosomal and digestive enzymes. Immunolocalization studies performed at the electron microscope level of resolution indicated that TAP and cathepsin B were both contained within the same cytoplasmic vacuoles at the earliest times after the onset of supramaximal secretagogue stimulation (Hofbauer et al, 1998b). Thus, as predicted by the co-localization hypothesis, trypsinogen activation appears to occur within the vacuoles which contain co-localized lysosomal hydrolases and digestive zymogens in this model. Role of cathepsin B Attempts to abort intra-pancreatic activation of trypsinogen and the development of pancreatitis by administration of cathepsin B inhibitors to intact animals were unsuccessful because complete inhibition of pancreatic cathepsin B cannot be achieved in vivo (Saluja et al, 1991). To overcome this problem, we established an in vitro model to replicate the events in secretagogue-induced pancreatitis. We found that intra-
Figure 5. Time course of caerulein-induced activation of trypsinogen. Rat pancreatic acini were incubated with either buffer alone (filled circles) or buffer containing 0.1 µM cerulein (filled squares) for varying times, and the trypsin activity in the homogenized acini was measured. Results are expressed as a percentage of the maximal activity obtained in each experiment. Values shown are expressed as means ± SEM obtained from at least four experiments. *P <0.05 compared with time 0. Reprinted from Saluja et al (1997) with permission.
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acinar cell activation of trypsinogen can be detected when freshly prepared but otherwise normal pancreatic acini are incubated for 15–30 minutes in vitro with a supramaximally stimulating concentration of caerulein (Figure 5) and that activation can be prevented by inclusion of the cathepsin B inhibitor E-64D in the incubation mixture (Figure 6) (Saluja et al, 1997). Thus, as predicted by the co-localization hypothesis, complete inhibition of cathepsin B can prevent trypsinogen activation.
120
Enzyme activity (% of maximal)
100
80
60
40
20 (0)
0 BASAL
CER
CER + 1mM E-64d
Figure 6. Effect of E-64d on cathepsin B activity and caerulein (CER)-induced activation of trypsinogen. After a 30-minute pre-incubation with 1 mM cathepsin B inhibitor E-64d, rat pancreatic acini were further incubated with 0.1 µM CER together with E-64d for 30 minutes, and cathepsin B (hatched bars) and trypsin (filled bars) activities in the homogenized acini were measured. Results are expressed as a percentage of the activity obtained after incubation with CER alone. Values shown are means ± SEM obtained from at least three experiments. Reprinted from Saluja et al (1997, Gastroenterology 113: 304–310) with permission.
Cell injury The in vitro system that involves incubating freshly prepared rat pancreatic acini with a supramaximally stimulating concentration of caerulein was also used to evaluate the relationship between intra-acinar cell activation of trypsinogen and acinar cell injury. Trypsinogen activation was found to be dependent upon a secretagogue-induced rise in intracellular ionized calcium and to precede evidence of cell injury. Prevention of secretagogue-induced trypsinogen activation or, alternatively, inhibition of trypsin activity, was found to prevent acinar cell injury (Saluja et al, unpublished observations). These observations, therefore, support the co-localization hypothesis by demonstrating that the prevention of cathepsin B-mediated trypsinogen activation and/or inhibition of active trypsin within acinar cells can prevent secretagogue-induced cell injury.
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DETERMINANTS OF SEVERITY The studies summarized above support the hypothesis that the earliest changes in pancreatitis occur within acinar cell cytoplasmic vacuoles where the co-localization of digestive enzyme zymogens with lysosomal hydrolases leads to trypsinogen activation, activation of the other zymogens, and ultimately to acinar cell injury. The colocalization phenomenon and zymogen activation are common features shared by many of the models of pancreatitis, yet the severity of these models varies considerably. Similarly, clinical pancreatitis can be either mild or severe. This had suggested that events which occur subsequent to intra-acinar cell activation of zymogens might play an important role in regulating the severity of pancreatitis. Apoptosis/necrosis Cell death may occur as a result of either apoptosis or necrosis. The former is an energy dependent, genetically programmed process which usually involves little or no inflammation. In contrast, necrosis is usually accompanied by an extensive inflammatory reaction. The events which underlie pancreatic acinar cell apoptosis have not been extensively characterized but studies to date indicate that it is, at least in part, triggered by tumour necrosis factor-α, generated by neutrophils and, perhaps, by acinar cells themselves (Gukovskaya et al, 1997). As in other types of cell, apoptosis in acinar cells involves the internucleosomal degradation of genomic DNA catalysed by a Ca2+-Mg2+-dependent endonuclease. We (Kaiser et al, 1995) and others (Gukovskaya et al, 1997) have found that extensive apoptosis of acinar cells occurs in models of mild pancreatitis, whereas acinar cell necrosis is a characteristic of severe pancreatitis. Furthermore, interventions that promote apoptosis lead to a reduction in the severity of pancreatitis (Saluja et al, 1996; Bhatia et al, 1998b) while those that prevent apoptosis cause a worsening of pancreatitis (Kaiser et al, 1996). This has suggested that apoptosis might be a teleologically favourable response to acinar cell injury in pancreatitis and that therapeutic interventions designed to promote acinar cell apoptosis might prove to be beneficial in the management of patients with clinical pancreatitis. Mediators of inflammation The local as well as the systemic changes of pancreatitis are closely regulated by a vast array of factors which co-ordinate and control the inflammatory response. Considerable recent attention has been directed at identifying and characterizing those factors, but our knowledge concerning this complex process remains very limited. Until recently most studies in this area were performed using inhibitors designed to block the effects of putative pro-inflammatory factors. More recently, targeted gene deletion has been employed to generate knock-out mice which lack receptors specific for those factors or which cannot generate the factors themselves. Using these, as well as other methods, Norman and co-workers (Denham et al, 1997) as well as Pandol and his colleagues (Gukovskaya et al, 1997) have shown that inteleukin-1 (IL-1) and TNF-α play important pro-inflammatory roles in pancreatitis. Reber and his group (Rongione et al, 1997) as well as van Laethem et al (1995) have shown that IL-10 exerts a potent anti-inflammatory influence in pancreatitis. We (Hofbauer et al, in press) and others (Formela et al, 1994) have found that platelet activating factor (PAF) has a proinflammatory effect in experimental pancreatitis and that either inhibition of PAF
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action or hydrolysis of PAF can protect against pancreatitis and pancreatitis-associated lung injury. The neurokinin substance P, acting via neurokinin-1 receptors (NK-1r), which normally functions as a neuraltransmitter in afferent vagal fibres from the pancreas, has been found to exert a potent pro-inflammatory effect in pancreatitis, mediating so-called neurogenic inflammation in the pancreas and pancreatitisassociated lung injury (Bhatia et al, 1998a) (Figure 7). Chemokines also play important roles in regulating the local and systemic changes in pancreatitis by controlling the activation and recruitment of inflammatory cells. Mice which lack the C-C chemokine receptor CCR-1 are protected against pancreatitis-associated lung injury (Figure 8) (Gerard et al, 1997). Finally, complement and the factors generated during complement activation are believed to play important roles in pancreatitis. The nature of that role, however, is far from clear as shown by our recent observation that the anaphylatoxin C5a, which would be expected to play a pro-inflammatory role in pancreatitis, actually has a dramatic anti-inflammatory effect (Bhatia et al, unpublished observations).
Figure 7. Effects of NK1R deletion on pancreatitis-associated lung injury. Mice were given 12 hourly injections of caerulein (50 µg/kg intraperitoneally). One hour after the last injection of caerulein, mice were killed and the capillary leakage of FITC-albumin and lung MPO activity were measured. Values are expressed as a percentage of the value obtained for wild-type animals given caerulein. These values (100%) were as follows: lung MPO activity: 3.06 ± 0.37; FITC-albumin bronchoalveolar lavage fluid to serum ratio 2.13 ± 0.24. Results shown are the mean ± SEM for 10 or more animals in each group. Asterisks indicate P < 0.05 when NK1R–/– animals were compared with NK1R+/+ animals. Reprinted from Bhatia et al (1998, Proceedings of the National Academy of Sciences of the USA 95: 4760–4765) with permission.
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Figure 8. Effects of CCR1 receptor deletion on pancreatitis-associated lung injury. Capillary leakage of FITC-albumin and lung MPO activity were measured as described in the text. Solid bars indicate wild-type control animals, open bars indicate CCR1 receptor deficient control animals, single-hatched bars indicate wild-type animals given 12 hourly injections of a supramaximally stimulating dose of caerulein, and doublehatched bars indicate CCR1 receptor-deficient animals given 12 hourly injections of caerulein. Values are expressed as a percentage of the value obtained for wild-type animals given caerulein. Results shown are mean ± SEM values for 10 or more animals in each group. *P <0.05 when CCR1 receptor-deficient animals were compared with wild-type animals. Reprinted from Gerard et al (1997, Journal of Clinical Investigation 100: 2022–2027) with permission.
CONCLUSION Acute pancreatitis is a complex disease which can be initiated by a variety of events. The earliest changes in pancreatitis occur within acinar cells where activation of digestive enzyme zymogens leads to acinar cell injury. Subsequent events, occurring as a result of acinar cell injury, regulate the severity of pancreatitis and the systemic response, including the associated lung injury. Future studies are likely to broaden our understanding of these various processes and, in this way, suggest therapies which might either abort or reduce the severity of clinical pancreatitis. REFERENCES *Bhatia M, Saluja AK, Hofbauer B et al (1998a) Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury. Proceedings of the National Academy of Sciences of the USA 95: 4760–4765. Bhatia M, Wallig M, Hofbauer B et al (1998b) Induction of apoptosis in pancreatic acinar cells reduces the severity of acute pancreatitis. Biochemical and Biophysical Research Communications 246: 476–483. Denham W, Yang J, Fink G et al (1997) Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis. Gastroenterology 113: 1741–1746. Figarella C, Miszczuk-Jamska & Barrett AL (1998) Possible lusosomal activation of pancreatic zymogens: activation of both human trypsinogen by cathepsin B and spontaneous acid activation of human trypsinogen 1. Hoppe Seyler’s Journal of Biological Chemistry 369: 293–298.
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