Bernd Hofbauer

The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury.

BACKGROUND & AIMS Intercellular adhesion molecule 1 (ICAM-1) and neutrophils play important roles in many inflammatory processes, but their importance in both acute pancreatitis and pancreatitis-associated lung injury has not been defined. METHODS To address this issue, mice that do not express ICAM-1 were used and depleted of neutrophils by administration of antineutrophil serum. Pancreatitis was induced by administering either supramaximal doses of the secretagogue cerulein or feeding a choline-deficient, ethionine-supplemented diet. The severity of pancreatitis was evaluated by quantitating serum amylase, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (i.e., neutrophil content). Lung injury was evaluated by quantitating lung myeloperoxidase activity and pulmonary microvascular permeability. ICAM-1 was quantitated by enzyme-linked immunosorbent assay and was localized by light-microscopic immunohistochemistry. RESULTS It was found that serum, pancreas, and lung ICAM-1 levels increase during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in ICAM-1. Neutrophil depletion also reduces the severity of both pancreatitis and lung injury. However, the combination of neutrophil depletion with ICAM-1 deficiency does not reduce the severity of pancreatitis or lung injury to a greater extent than either neutrophil depletion or ICAM-1 deficiency alone. Neither pancreatitis nor pancreatitis-associated lung injury are completely prevented by ICAM-1 deficiency, neutrophil depletion, or combined ICAM-1 deficiency plus neutrophil depletion. CONCLUSIONS The observations indicate that ICAM-1 plays an important, neutrophil-mediated, proinflammatory role in pancreatitis and pancreatitis-associated lung injury. The studies also indicate that ICAM-1 and neutrophil-independent events also contribute to the evolution of pancreatitis and lung injury in these models.

Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B

BACKGROUND & AIMS One of the central, unresolved issues in the pathogenesis of acute pancreatitis is the uncertainty regarding the mechanisms responsible for the premature intrapancreatic activation of digestive enzyme zymogens. The aim of the current study was to develop and characterize an in vitro system that might mimic the events leading to trypsinogen activation within the pancreas during pancreatitis. METHODS Activation of trypsinogen in response to stimulation with cerulein was quantitated in isolated rat pancreatic acini. RESULTS Activation of trypsinogen was detected within 10 minutes of exposing isolated rat pancreatic acini, in Ca2+-containing buffer, to a supramaximally stimulating concentration of cerulein in vitro. Complete inhibition of pancreatic cathepsin B activity with E-64d, a specific, potent and irreversible cathepsin B inhibitor, prevents cerulein-induced in vitro trypsinogen activation. CONCLUSIONS In vitro activation of trypsinogen can be detected when pancreatic acini are exposed to a supramaximally stimulating dose of cerulein. The results using this in vitro system support the hypothesis that the appearance of active trypsin within the pancreas during the early stages of cerulein-induced pancreatitis reflects activation of trypsinogen by the lysosomal hydrolase cathepsin B.

Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats

Supramaximal stimulation of the pancreas with the CCK analog caerulein causes acute edematous pancreatitis. In this model, active trypsin can be detected in the pancreas shortly after the start of supramaximal stimulation. Incubation of pancreatic acini in vitro with a supramaximally stimulating caerulein concentration also results in rapid activation of trypsinogen. In the current study, we have used the techniques of subcellular fractionation and both light and electron microscopy immunolocalization to identify the site of trypsinogen activation and the subsequent fate of trypsin during caerulein-induced pancreatitis. We report that trypsin activity and trypsinogen-activation peptide (TAP), which is released on activation of trypsinogen, are first detectable in a heavy subcellular fraction. This fraction is enriched in digestive enzyme zymogens and lysosomal hydrolases. Subsequent to trypsinogen activation, both trypsin activity and TAP move to a soluble compartment. Immunolocalization studies indicate that trypsinogen activation occurs in cytoplasmic vacuoles that contain the lysosomal hydrolase cathepsin B. These observations suggest that, during the early stages of pancreatitis, trypsinogen is activated in subcellular organelles containing colocalized digestive enzyme zymogens and lysosomal hydrolases and that, subsequent to its activation, trypsin is released into the cytosol.Supramaximal stimulation of the pancreas with the CCK analog caerulein causes acute edematous pancreatitis. In this model, active trypsin can be detected in the pancreas shortly after the start of supramaximal stimulation. Incubation of pancreatic acini in vitro with a supramaximally stimulating caerulein concentration also results in rapid activation of trypsinogen. In the current study, we have used the techniques of subcellular fractionation and both light and electron microscopy immunolocalization to identify the site of trypsinogen activation and the subsequent fate of trypsin during caerulein-induced pancreatitis. We report that trypsin activity and trypsinogen-activation peptide (TAP), which is released on activation of trypsinogen, are first detectable in a heavy subcellular fraction. This fraction is enriched in digestive enzyme zymogens and lysosomal hydrolases. Subsequent to trypsinogen activation, both trypsin activity and TAP move to a soluble compartment. Immunolocalization studies indicate that trypsinogen activation occurs in cytoplasmic vacuoles that contain the lysosomal hydrolase cathepsin B. These observations suggest that, during the early stages of pancreatitis, trypsinogen is activated in subcellular organelles containing colocalized digestive enzyme zymogens and lysosomal hydrolases and that, subsequent to its activation, trypsin is released into the cytosol.

The effects of neutrophil depletion on a completely noninvasive model of acute pancreatitis-associated lung injury

SummaryConclusionA completely noninvasive animal model of acute pancreatitis-associated lung injury was used to show that neutrophils, activated by pancreatitis, play a key role in mediating pancreatitis-associated lung injury.BackgroundSignificant pulmonary complications have been known to occur in over 50% of patients with severe acute pancreatitis. Recent studies using a variety of animal models of pancreatitis have suggested that neutrophil activation may play an important role in mediating lung injury. However, in these models, the interpretation of the results is complicated because surgical manipulations alone could have resulted in the activation of neutrophils.MethodsYoung female mice were fed a choline deficient ethionine (CDE) supplemented diet. The severity of pancreatitis was evaluated by measuring hyperamylasemia, acinar cell necrosis, and pancreatic myeloperoxidase activity. Lung injury was quantified by measuring lung microvascular permeability and lung myeloperoxidase activity. To evaluate the role of neutrophils in CDE diet-induced pancreatitis-associated lung injury, animals were pretreated with antineutrophil serum.ResultsMice fed the CDE diet develop pancreatitis-associated lung injury. Pretreatment of mice with antineutrophil serum results in marked depletion of circulating neutrophils. Under these conditions, the severity of pancreatitis is reduced and lung injury is completely prevented.

Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis

BACKGROUND & AIMS Recent reports suggest that platelet-activating factor (PAF) plays a role in pancreatitis and pancreatitis-associated lung injury. In this study, the effects on these processes of termination of PAF action by recombinant PAF-acetylhydrolase (rPAF-AH) were investigated. METHODS Rats were given rPAF-AH and then infused with a supramaximally stimulating dose of cerulein to induce mild pancreatitis. Opossums underwent biliopancreatic duct ligation to induce severe pancreatitis, and rPAF-AH administration was begun 2 days later. RESULTS In mild, secretagogue-induced pancreatitis, rPAF-AH given before the cerulein reduced hyperamylasemia, acinar cell vacuolization, and pancreatic inflammation but did not alter pancreatic edema or pulmonary microvascular permeability. In severe, biliopancreatic duct ligation-induced pancreatitis, rPAF-AH delayed and reduced the extent of inflammation and acinar cell injury/necrosis and completely prevented lung injury even though the rPAF-AH administration was begun after the onset of pancreatitis. CONCLUSIONS PAF plays an important role in the regulation of pancreatic injury but not pancreatic edema or increased pulmonary microvascular permeability in mild, secretagogue-induced pancreatitis. PAF plays a critical role in the regulation of progression of pancreatic injury and mediation of pancreatitis-associated lung injury in severe biliary pancreatitis. Amelioration of pancreatitis and prevention of pancreatitis-associated lung injury can be achieved with rPAF-AH even if treatment is begun after pancreatitis is established.

A cholecystokinin-releasing factor mediates ethanol-induced stimulation of rat pancreatic secretion.

The mechanisms by which short-term ethanol administration alters pancreatic exocrine function are unknown. We have evaluated the effects of ethanol administration on pancreatic secretion of digestive enzymes. In our studies, anesthetized as well as conscious rats were given ethanol at a rate sufficient to cause the blood ethanol concentration to reach levels associated with clinical intoxication. Ethanol was administered over a 2-h period during which blood ethanol levels remained stably elevated. We report that intravenous administration of ethanol results in a transient increase in pancreatic amylase output and plasma cholecystokinin (CCK) levels. The ethanol-induced increase in amylase output can be completely inhibited by the CCK-A receptor antagonist L-364,718 and partially inhibited by the muscarinic cholinergic antagonist atropine. The ethanol-induced rise in amylase output can be completely prevented by instillation of trypsin into the duodenum or by lavage of the duodenum with saline during ethanol administration. Furthermore, the intraduodenal activity of a CCK-releasing factor is increased by infusion of ethanol. These studies indicate that administration of ethanol causes rat pancreatic exocrine secretion to increase. This phenomenon is mediated by a trypsin-sensitive CCK-releasing factor which is present within the duodenal lumen. These observations lead us to speculate that repeated CCK-mediated ethanol-induced stimulation of pancreatic digestive enzyme secretion may play a role in the events which link ethanol abuse to the development of pancreatic injury.