Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis

Abstract

In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.

Figure 1

Cerulein-stimulated in vitro amylase secretion from pancreatic acini. Acini were prepared from both wild-type (□) and PI3Kγ-deficient animals (•) and incubated with varying concentrations of cerulein for 30 minutes. Amylase release was quantitated as described in Materials and Methods. Results represent mean ± SEM for three animals in each group.

Figure 2

Effects of PI3Kγ deletion on cerulein-induced acute pancreatitis. Mice were administered six intraperitoneal injections of cerulein and killed 1 hour after the last injection of cerulein. A: Representative micrographs of morphological alterations induced in exocrine pancreas by 6 hours of cerulein administration (cerulein 6 hours) compared with 6 hours of saline administration (control), in wild-type (WT) and PI3Kγ-deficient (PI3Kγ−/−) mice. B: Quantitation of cerulein-induced pancreatic injury. Pancreatic water content, serum amylase activity, and acinar cell injury/necrosis were measured as described in the text. Wild-type mice, open columns; PI3Kγ-deficient mice, shaded columns. Results are mean ± SEM values for five or more animals in each group. *, P < 0.05 versus control. C: Effects of PI3Kγ deletion on acinar cell apoptosis in cerulein-induced acute pancreatitis. Apoptotic acinar cells were detected by TUNEL as described in Materials and Methods. Wild-type mice (□) and PI3Kγ-deficient mice (•) were administered six intraperitoneal injections of cerulein and killed 1 hour after the last injection of cerulein. *, P < 0.05 PI3Kγ-deficient versus wild-type mice. D: Immunohistochemical analysis of caspase-3 activity in exocrine pancreas from wild-type (WT) and PI3Kγ-deficient (PI3Kγ−/−) mice after 6 hours of cerulein administration. Original magnifications: ×400 (A); ×1000 (D).

Figure 3

Effects of PI3Kγ deletion on cerulein-induced acute pancreatitis. Mice were administered 13 intraperitoneal injections of cerulein and killed 1 hour after the last injection of cerulein. A: Representative micrographs of morphological alterations induced in exocrine pancreas by 13 hours of cerulein administration (cerulein 13h) compared with 13 hours of saline administration (Control), in wild-type (WT) and PI3Kγ-deficient (PI3Kγ−/−) mice. Thirteen hours of cerulein administration induced evident morphological alterations and massive neutrophil infiltration of the exocrine pancreas compared with controls. Foci of coagulative necrosis are also evident. Original magnifications, ×400. B: Quantitation of cerulein-induced pancreatic injury. Serum amylase activity, acinar cell injury/necrosis, and neutrophil infiltration were measured as described in the text. Wild-type mice, open columns; PI3Kγ-deficient mice, shaded columns. Results are mean ± SEM values for five or more animals in each group. *, P < 0.05 versus control. C: Effects of PI3Kγ deletion on pancreatic COX-2 expression during cerulein-induced acute pancreatitis. Pancreata from wild-type (WT) and PI3Kγ-deficient (PI3Kγ−/−) mice were harvested after 13 hours from the beginning of saline (C) or cerulein (Cer) administration, and analyzed by Western blot for COX-2 and α-tubulin expression, as described in Materials and Methods.

Figure 4

Effects of PI3Kγ deletion on CDE diet-induced acute pancreatitis. Young female mice were fasted for 24 hours and then fed the CDE diet for 48 hours, as described in Materials and Methods. A: Representative micrographs of morphological alterations of exocrine pancreas (top) and lung (bottom) after CDE diet administration in wild-type (WT) and PI3Kγ-deficient (PI3Kγ−/−) mice. Original magnifications, ×400. B: Quantitation of CDE diet-induced pancreatic injury. Pancreatic water content, acinar cell injury/necrosis, and neutrophil infiltration were measured as described in the text. Wild-type mice, open columns; PI3Kγ-deficient mice, shaded columns. Results are mean ± SEM values for five or more animals in each group. *, P < 0.05 versus control. C: Quantitation of CDE diet-induced lung injury. The extent of alveolar-capillary membrane thickening and of neutrophil infiltration were measured as described in the text. Wild-type mice, open columns; PI3Kγ-deficient mice, shaded columns. Results are mean ± SEM values for five or more animals in each group. *, P < 0.05 versus control.

Figure 5

Mortality curve for wild-type (□) and PI3Kγ-deficient animals (•) during CDE diet-induced acute pancreatitis. Young female mice were fed a CDE diet for 48 hours and then regular chow for the following days, and mortality was recorded, as described in Materials and Methods. P < 0.03 for PI3Kγ-deficient mice versus wild-type mice.