Volume 129, Issue 2, Pages 665-681 (August 2005) A Proinflammatory, Antiapoptotic Phenotype Underlies the Susceptibility to Acute Pancreatitis in Cystic Fibrosis Transmembrane Regulator (−/−) Mice  Matthew J. Dimagno, Sae-Hong Lee, Yibai Hao, Shi-Yi Zhou, Barbara J. McKenna, Chung Owyang  Gastroenterology  Volume 129, Issue 2, Pages 665-681 (August 2005) DOI: 10.1053/j.gastro.2005.05.059 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Baseline pancreas: cftr (-/-) vs WT mRNA gene expression of inflammatory mediators. Cftr (−/−) mice show a baseline mRNA overexpression of proinflammatory mediators in the pancreas. Whole-pancreas mRNA was isolated and reverse-transcribed, and gene transcripts were quantitated by real-time PCR (described in Methods) for the adhesion molecule ICAM-1, the proinflammatory cytokines TNF-α, IL-6, and IL-1β, the chemokine KC, and the anti-inflammatory mediators MCP-1 and IL-10. Data are expressed as -fold change compared with WT, which was assigned the value 1. Values were normalized to the GAPDH housekeeping gene [averaging 1.4-fold greater in cftr (−/−) mice compared with WT]. Columns represent the means and SEM (n = 6 per group). +P < .05 vs WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Effect of CFTR genotype on pancreatic histology at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg or saline and were killed 1 hour after 12 injections. H&E-stained sections were examined by light microscopy (200× magnification) for cerulein-injected (n = 5) and saline-injected (n = 4) groups. Images are representative of saline-injected (A) WT and (B) cftr (−/−) groups and cerulein-injected (C) WT and (D) cftr (−/−) groups. Morphological changes were graded 0–4 for edema, inflammation, and acinar cell injury or death, as described by Rongione et al.35 The individual histological score for each morphological criterion is provided for a representative section from the cerulein-injected groups (C and D): total—(C) 5 and (D) 7; edema—(C) 2 and (D) 3; inflammation—(C) 2 and (D) 3; acinar cell injury or death—(C) 1 and (D) 1. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Histologic scoring of acute pancreatitis at 12 hours. Effect of CFTR genotype on pancreatic inflammatory changes at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg or saline and were killed 1 hour after 12 injections. H&E-stained sections (see Figure 2) were examined by light microscopy (200× magnification) for cerulein-injected (n = 5) and saline-injected (n = 4) groups. (A) The composite severity score was based on morphological changes graded 0–4 for (B) edema, (C) inflammation, and (D) acinar cell injury or death, as described by Rongione et al.35 Columns represent the means and SEM. *P < .05 vs saline-injected controls; +P < .05 vs cerulein-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Nonhistological tissue quantitation of pancreatic edema and neutrophil infiltration at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. (A) Relative pancreatic water content in percentage (n = 4) and (B) MPO activity (mU/mg tissue weight; n = 4) were measured (described in Methods). Columns represent the means and SEM. **P < .0001 vs saline-injected controls; ++P < .005 vs cerulein-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Messenger RNA expression of (A) housekeeping genes and (B) neutrophil markers at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. Pancreas mRNA gene expression was quantitated by real-time PCR (as described in Figure 1). (A) Individual housekeeping genes were quantitated in WT mice (n = 4 per group) and expressed as -fold change relative to the saline-injected group. (B) The neutrophil mRNA markers neutrophil elastase and cathepsin G were quantitated in cftr (−/−) and WT mice (n = 3–6 per group) and expressed as -fold change relative to the saline-injected WT group, which was assigned the value 1. Values were normalized by using the ribosomal highly basic 23-kilodalton housekeeping gene (RB; validated in A) as the internal control gene. Columns represent the means and SEM. *P < .05 vs saline-injected control; **P < .005 vs saline-injected control; ***P < .0005 vs saline-injected control; +P < .05 vs cerulein-injected WT; ++P < .005 vs cerulein-injected WT; +++P < .05 vs saline-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Acute pancreatits at 12 hours cftr (-/-) vs WT mRNA gene expression. Messenger RNA expression of proinflammatory mediators at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. Pancreas mRNA gene expression was quantitated by real-time PCR (as described in Figure 1) for the adhesion molecule ICAM-1, the proinflammatory cytokines TNF-α, IL-6, and IL-1β, the chemokine KC, and the anti-inflammatory mediators monocyte chemotactic protein 1 and IL-10. Data were expressed as -fold change relative to the saline-injected WT, which was assigned the value 1. Values were normalized by using the ribosomal highly basic 23 kDa housekeeping gene (RB; validated in Figure 5A) as the internal control gene. The cftr (−/−) group had greater expression of proinflammatory mediators compared with WT after the induction of pancreatitis (analysis of variance; P < .005). Columns represent the means and SEM (n = 3 per group). *P < .05 vs saline-injected control; **P < .005 vs saline-injected control; ***P < .0005 vs saline-injected control; +P < .05 vs cerulein-injected WT; ++P < .005 vs cerulein-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 (A) DNA fragmentation and (B) cleavage of PARP protein. Reduced apoptosis in cftr (−/−) mouse pancreas at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. (A) Total DNA was isolated from whole pancreas, and DNA fragments were electrophoretically separated (described in Methods). (B) Whole-pancreas lysates were subjected to Western blotting studies (described in Methods) to detect changes in endogenous PARP (a 116-kilodalton protein) and the cleaved catalytic domain (89 kilodaltons). Relative optical densities for protein data were expressed as percentage of cerulein-injected WT and represent the means and SEM of saline-injected (n = 3) and cerulein-injected (n = 6) groups. +P < .001 vs cerulein-injected WT; *P < .0005 vs saline-injected control. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Reduced caspase 3–positive acinar cells (and apoptosis) in (A and B) cftr (−/−) mouse pancreas vs (C and D) WT at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. Caspase 3–stained sections were examined by immunofluorescence microscopy, and staining appeared as a red granular pattern within the cytoplasm of acinar cells (all images are at 600× magnification). The cerulein-injected cftr (−/−) group had infrequent acinar caspase 3 staining indicated by (A) immunofluorescence and had a significant loss of tissue architecture, as observed in the (B) overlay on the respective phase contrast (Nomarski differential interference contrast image). In comparison, the cerulein-injected WT group had significantly greater acinar caspase 3 activity, indicated by (C) immunofluorescent staining of multiple acinar cells, and had better preservation of tissue architecture, indicated by the (D) overlay images. Similar to the cerulein-injected cftr (−/−) group, minimal staining was detected in saline-injected cftr (−/−) or (+/+) mouse pancreas (not shown), and no staining was detected in negative controls (not shown), in which the primary caspase 3 antibody was omitted (bar = 10 μm). KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Quantitation of caspase 3–positive acinar cells (apoptosis) at 12 hours during acute pancreatitis. Mice received hourly IP injections of cerulein 50 μg/kg and were killed 1 hour after 12 injections. Immunofluorescent detection of active (cleaved) caspase 3 (see Figure 8) was expressed as positive (fluorescent) cells per field (400× magnification). Columns represent the means and SEM of saline-injected (n = 4) and cerulein-injected (n = 5) groups. *P = .0012 vs saline-injected controls; +P < .001 vs cerulein-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Effect of the cftr (−/−) genotype on pancreatic trypsin and serum lipase activities at 30 minutes and 12 hours of cerulein-induced acute pancreatitis. Mice received hourly IP injections of cerulein (50 μg/kg) and were killed 30 minutes after 1 injection or 1 hour after 12 hourly injections. Pancreatic trypsin and serum lipase activities were measured at (A and B) 30 minutes and (C and D) 12 hours. Basal serum lipase values averaged 109.4 ± 10.7 U/L for the WT and 76.6 ± 6.9 U/L for the cftr (−/−) groups (P < .05), and basal pancreatic trypsin activity averaged 0.35 ± 0.05 ng/mg protein for WT mice and 0.38 ± 0.03 ng/mg protein for cftr (−/−) groups. At the 12-hour pancreatitis time point (E), pancreatic trypsinogen content was determined enzymatically (as described in Methods) and used to (F) normalize trypsin activity, expressed as percentage trypsinogen content. Columns represent the means and SEM of saline-injected (n = 3–6) and cerulein-injected (n = 4–6) groups. *P < .05 vs saline-injected controls; +P < .05 vs cerulein-injected WT; ++P < .001 vs cerulein-injected WT; +++P < .05 vs saline-injected WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 11 Baseline pancreas: cftr (-/-) vs WT pancreatic digestive enzyme expression. Reduced constitutive pancreatic digestive enzyme (A) protein and (B) mRNA content in cftr (−/−) pancreas (filled boxes) compared with WT (open boxes). (A) Whole-pancreas lysates were subjected to Western blotting (described in Methods) to detect changes in baseline amylase, lipase, proelastase, and trypsinogen. Relative optical densities for protein data are expressed as percentage WT (n = 6) and represent the means and SEM. (B) Pancreas mRNA gene expression was quantitated by real-time PCR (as described in Figure 1) for the digestive enzymes amylase 2, lipase 1, proelastase 1, and trypsinogen 3. Data are expressed as -fold change compared with WT (n = 6 per group), which was assigned the value 1. Values were normalized to the GAPDH housekeeping gene [averaging 1.4-fold greater in cftr (−/−) mice compared with WT]. Columns represent the means and SEM (n = 6 per group). +P < .05 vs WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 12 Reduced in vivo stimulated pancreatic secretory response in cftr (−/−) mice. Bile-pancreatic juice was collected every 15 minutes, basal output was determined from 15 to 30 minutes, and intravenous CCK-8 was infused starting at 30 minutes and ending at 90 minutes (described in Methods). Stimulated bile-pancreatic protein output is expressed as micrograms per minute. Data points are shown as flat lines and reflect sample collection over 15-minute intervals. Statistical analysis was performed on the CCK-8–dependent pancreatic protein secretion (upper right panel) determined by subtracting the basal from the pooled peak secretagogue response between 45 and 75 minutes. Data are the means and SEM of 5–9 mice per group. +P < .0005 vs CCK-8–treated WT. KO, knockout. Gastroenterology 2005 129, 665-681DOI: (10.1053/j.gastro.2005.05.059) Copyright © 2005 American Gastroenterological Association Terms and Conditions