Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis

doi:10.1053/gast.2002.32409

Gastroenterology

Volume 122, Issue 4, April 2002, Pages 974-984

https://doi.org/10.1053/gast.2002.32409 Get rights and content

Abstract

Background & Aims: Intrapancreatic activation of digestive enzymes is a key event in the parenchymal cell injury of pancreatitis. We hypothesized that neutrophils recruited to the pancreas during pancreatitis may contribute to such activation. Methods: To cause experimental pancreatitis, rats and mice were treated with high doses of cerulein. Activation of the digestive enzyme, trypsin, was measured in pancreatic homogenates using a fluorogenic assay and localized immunocytochemically with antibody to trypsin-activation peptide (TAP). Results: Compared with controls, rats depleted of neutrophils with antineutrophil serum exhibited a marked attenuation in intrapancreatic trypsin activation and acinar cell TAP labeling induced by high-dose cerulein. To examine the mechanism, mice deficient in either nicontinamide adenine dinucleotide phosphate (NADPH) oxidase, or myeloperoxidase (MPO) were studied for trypsin activation. Mice deficient in NADPH oxidase exhibited attenuation of the cerulein-induced trypsin activation, but those deficient in MPO did not. Using measurements of Western blot analysis, generation of reactive oxygen species, and immunocytochemistry, we demonstrated the NADPH oxidase activity is in neutrophils and not pancreatic acinar tissue. Conclusions: The results demonstrate a novel role for neutrophils infiltrating the pancreas in pathologic activation of digestive enzymes in acute pancreatitis and indicate that this effect is mediated by products of NADPH oxidase.

GASTROENTEROLOGY 2002;122:974-984

Section snippets

Animals

For the experiments, we used Sprague-Dawley rats weighing 250–300 g. Mice deficient in NADPH oxidase, p47 (phox −/−), were described previously.²² These mice and their wild-type controls were both BALB/c mice. Mice deficient in MPO were generated using standard gene-targeting techniques and verified to lack MPO based on the absence of peroxidase and chlorination activity in mouse neutrophils.²³ These mice and their wild-type controls were both on the C57BL/6 background.

Neutrophil depletion of rats

Neutrophil depletion was

Results

Pretreatment of rats with ANS resulted in a marked decrease in circulating neutrophils at 6 hours after administration. Circulating neutrophils were 2000 ± 250/mL (n = 5) in untreated rats and 38 ± 42/mL (n = 18) in ANS-treated rats. Cerulein infusions were initiated 6 hours after ANS administration.

We first determined the effects of neutrophil depletion on pancreatic trypsin activity in rats infused with high-dose cerulein (Figure 1).

. Neutrophil depletion of rats results in an attenuation of

Discussion

Taken together, the results of the present study indicate that neutrophils infiltrating the pancreas facilitate intrapancreatic trypsin activation during cerulein-induced experimental pancreatitis. Immunocytochemical localization experiments using specific antibodies to TAP indicate that the trypsin activation mediated by neutrophils occurs within the pancreatic acinar cell. Finally, the results with the NADPH oxidase-deficient and MPO-deficient mice suggest that NADPH oxidase products (e.g.,

Acknowledgements

The authors thank Dr. Ilya Gukovsky for advice and discussion, Michelle Mouria and Shakti Khanna for help with measurements of trypsin activities and neutrophil counts, and Yoon Jung for help in preparing this manuscript.

References (57)

JL Frossard et al.
The role of intercellular adhesion molecule-1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury
Gastroenterology
(1999)
D Sandoval et al.
The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis
Gastroenterology
(1996)
SJ Pandol et al.
The agonist-sensitive calcium pool in the pancreatic acinar cell: activation of plasma membrane Ca 2+ influx mechanism
J Biol Chem
(1987)
SJ Pandol et al.
Ethanol diet increases the sensitivity of the rats to pancreatitis induced by cholecystokinin octapeptide
Gastroenterology
(1999)
C Dahlgren et al.
Respiratory burst in human neutrophils
J Immunol Methods
(1999)
RT Jensen et al.
Interaction of CCK with pancreatic acinar cells
Trends Pharmacol Sci
(1989)
BM. Babior
NADPH oxidase: an update
Blood
(1999)
KS Guice et al.
Superoxide dismutase and catalase: a possible role in established pancreatitis
Am J Surg
(1986)
Y Kikuchi et al.
Transgenic copper/zinc-superoxide dismutase ameliorates caerulein-induced pancreatitis in mice
Biochem Biophys Res Com
(1997)
C Niederau et al.
Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules
Free Radic Biol Med
(1996)

KJ. Davies

Protein damage and degradation by oxygen radicals. I. General principles

J Biol Chem

(1987)

SK Ray et al.

Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells

Brain Res

(1999)

I Ishihara et al.

Activation of calpain precedes morphological alterations during hydrogen peroxide-induced apoptosis in neuronally differentiated mouse embryonal carcinoma P19 cell line

Neurosci Lett

(2000)

M Topazian et al.

Acute pancreatitis

ML Steer et al.

The cell biology of experimental pancreatitis

N Engl J Med

(1987)

DC Whitcomb et al.

Hereditary pancreatitis is caused by a mutation on the cationic trypsinogen gene

Nat Genet

(1996)

R Luthen et al.

Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats

Am J Physiol

(1995)

SJ. Pandol

Pancreatic physiology

MC Geokas et al.

Free proteolytic enzymes in pancreatic juice of patients with acute pancreatitis

Am J Dig Dis

(1974)

KW Simpson et al.

Cholecystokinin-8 induces edematous pancreatitis in dogs associated with short burst of trypsinogen activation

Dig Dis Sci

(1995)

FS Gorelick et al.

Intracellular proteolysis of pancreatic zymogens

Yale J Biol Med

(1992)

M Katz et al.

Effect of ethanol on cholecystokinin-stimulated zymogen conversion in pancreatic acinar cells

Am J Physiol

(1996)

T Otani et al.

Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis

Am J Physiol

(1998)

B Hofbauer et al.

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

Am J Physiol

(1998)

T Grady et al.

Edema and intrapancreatic trypsinogen activation precede glutathione depletion during caerulein pancreatitis

Am J Physiol

(1996)

A Saluja et al.

Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis

Am J Physiol

(1987)

SD Leach et al.

Influence of chloroquine on diet-induced pancreatitis

Pancreas

(1993)

SD Leach et al.

Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin

J Clin Invest

(1991)

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^☆: Address requests for reprints to: Anna S. Gukovskaya, Ph.D., VA Greater Los Angeles Healthcare System, Building 258, Room 340, 11301 Wilshire Boulevard, Los Angeles, California 90073. e-mail: [email protected].

^☆☆: Supported by the Department of Veterans Affairs, the Research Center for Alcoholic Liver and Pancreatic Diseases (P50 AA11999) funded by the National Institute on Alcohol Abuse and Alcoholism, the Andrew Barnes Family Foundation, the National Institutes of Health grant HL30568 (to A.J.L.) and Societat Catalana de Digestologia, Spain (to E.V.).

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Basic ResearchNeutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis☆,☆☆

Abstract

Section snippets

Animals

Neutrophil depletion of rats

Results

Discussion

Acknowledgements

Gastroenterology

Gastroenterology

J Biol Chem

Gastroenterology

J Immunol Methods

Trends Pharmacol Sci

Blood

Am J Surg

Biochem Biophys Res Com

Free Radic Biol Med

J Biol Chem

Brain Res

Neurosci Lett

Acute pancreatitis

The cell biology of experimental pancreatitis

N Engl J Med

Hereditary pancreatitis is caused by a mutation on the cationic trypsinogen gene

Nat Genet

Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats

Am J Physiol

Pancreatic physiology

Free proteolytic enzymes in pancreatic juice of patients with acute pancreatitis

Am J Dig Dis

Cholecystokinin-8 induces edematous pancreatitis in dogs associated with short burst of trypsinogen activation

Dig Dis Sci

Intracellular proteolysis of pancreatic zymogens

Yale J Biol Med

Effect of ethanol on cholecystokinin-stimulated zymogen conversion in pancreatic acinar cells

Am J Physiol

Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis

Am J Physiol

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

Am J Physiol

Edema and intrapancreatic trypsinogen activation precede glutathione depletion during caerulein pancreatitis

Am J Physiol

Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis

Am J Physiol

Influence of chloroquine on diet-induced pancreatitis

Pancreas

Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin

J Clin Invest

Basic Research
Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis☆,☆☆