Volume 122, Issue 4, Pages 974-984 (April 2002) Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis  Anna S. Gukovskaya, Eva Vaquero, Vjekoslav Zaninovic, Fred S. Gorelick, Aldons J. Lusis, Marie–Luise Brennan, Steven Holland, Stephen J. Pandol  Gastroenterology  Volume 122, Issue 4, Pages 974-984 (April 2002) DOI: 10.1053/gast.2002.32409 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Neutrophil depletion of rats results in an attenuation of intrapancreatic trypsin activation induced by high-dose cerulein. Rats were treated with ANS or no ANS. Then, 6 hours later, the rats received infusion of 5 μg · kg−1 · h−1 cerulein (or saline) for the indicated time period. At the end of the infusion, the animals were killed, and activated trypsin level was measured as described in the Materials and Methods section. The values reported are means ± standard errors from 3 to 6 rats for each time point. Trypsin activity in saline-infused normal and ANS-treated rats is given at time point 0. This activity did not change with time, and ANS pretreatment had no significant effect on trypsin activity in saline-infused rats. An asterisk (*) indicates that the values for cerulein-infused rats treated with ANS were statistically significantly (P < 0.05) less (at the same time point) than those for cerulein-infused rats without ANS treatment. Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Neutrophil depletion of rats results in an attenuation of the cerulein-induced increases in serum amylase (A) and pancreatic neutrophil infiltration (B). Rats were treated with ANS or no ANS, and 6 hours later received high-dose cerulein (or saline) infusion for the indicated period. At the end of the infusion, the rats were killed, and serum amylase and pancreatic neutrophil levels were measured. The reported values are means ± standard error from 3 to 6 animals for each time point. The values for saline-infused rats are given at time point 0. An asterisk (*) indicates that the values for the cerulein-infused animals were significantly greater than those for the saline-infused animals (P < 0.05; Student t test). A pound sign (#) indicates that the values for cerulein-infused animals treated with ANS were statistically significantly (P < 0.05) less (at the same time point) than those for cerulein-infused animals without ANS treatment. Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Neutrophil depletion of rats results in an attenuation of cerulein-induced TAP immunoreactivity in pancreatic acinar cells. Rats received saline (A, D) or cerulein (B, E) infusion for 6 hours, or ANS pretreatment followed by cerulein infusion (C, F). After infusion, pancreata were removed and processed for TAP immunocytochemistry as described in the Materials and Methods section. (A–C) TAP immunofluorescence; (D–F) corresponding phase images. Note the intense TAP immunoreactivity in small vesicles (arrowheads) and larger vacuoles (arrow) within acinar cells after cerulein infusion of neutrophil-containing rats (B). Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Intrapancreatic trypsin, trypsinogen, neutrophil infiltration, and serum amylase in NADPH-oxidase deficient and wild-type mice treated with cerulein. Mice were treated with cerulein as described in the Materials and Methods section. After treatment, the pancreas and blood were taken for measurement of (A) trypsin, (B) trypsinogen, (C) neutrophil infiltration, and (D) serum amylase. Values are means ± standard errors from at least 4 animals in each group. Serum amylase levels are presented as ratios to those in saline-treated animals, which were 2200 ± 150 and 3200 ± 320 in saline-treated control and NADPH oxidase-deficient mice, respectively. An asterisk (*) indicates that the values for cerulein-treated mice were significantly greater than those for the control (saline-injected) mice (P < 0.05; Student t test). A pound sign (#) indicates that the values for cerulein-treated NADPH oxidase-deficient mice were statistically significantly (P < 0.05) less than those for wild-type mice treated with cerulein. Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Intrapancreatic trypsin in MPO-deficient and wild-type mice treated with cerulein. Mice were treated with cerulein as described in the Materials and Methods section. After treatment, the pancreas was taken for measurement of active trypsin. Values are means ± standard errors from at least 4 separate animals in each group. An asterisk (*) indicates that the values for cerulein-treated mice were significantly greater than those for the control (saline-injected) mice (P < 0.05; Student t test). Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 NADPH oxidase is present in neutrophils but not in pancreatic acinar cells. (A) Western blot analysis for NADPH oxidase in neutrophils and pancreatic acinar cells. Lysates from rat blood neutrophils and from pancreatic acinar cells, which were incubated with and without 0.1 μmol/L cerulein for 30 minutes, were probed with antibodies against NADPH oxidase subunits p47: whole molecule (a), p47 C-terminus (b), or p67 (c). (B) ROS production by rat blood neutrophils and pancreatic acinar cells. Neutrophils (a, b) were stimulated with 50 nmol/L phorbol myristate acetate and acinar cells (c, d) with 0.1 μmol/L CCK in the absence (a, c) or presence (b, d) of 10 μmol/L DPI, a specific inhibitor of NADPH oxidase. ROS production was measured by luminol-amplified chemiluminescence as described in the Materials and Methods section. The reaction was started by addition of stimuli. Data are representative of 3 independent experiments. (C) Immunofluorescence localization of p47 and p67 in pancreas from animals with cerulein pancreatitis: wild-type mice (a, b), p47-deficient mice (c, d, e), and rat (f). Pancreatic sections were stained with the antibody against p47 (a, c, f) or p67 (e) as described in the Materials and Methods section. To discriminate between neutrophils (arrows) and acinar cells, F-actin in the same sections was visualized by staining with rhodamine-conjugated phalloidin (b, d). Gastroenterology 2002 122, 974-984DOI: (10.1053/gast.2002.32409) Copyright © 2002 American Gastroenterological Association Terms and Conditions