Deborah E. Williard

The novel cytokine interleukin-33 activates acinar cell proinflammatory pathways and induces acute pancreatic inflammation in mice.

Background Acute pancreatitis is potentially fatal but treatment options are limited as disease pathogenesis is poorly understood. IL-33, a novel IL-1 cytokine family member, plays a role in various inflammatory conditions but its role in acute pancreatitis is not well understood. Specifically, whether pancreatic acinar cells produce IL-33 when stressed or respond to IL-33 stimulation, and whether IL-33 exacerbates acute pancreatic inflammation is unknown. Methods/Results In duct ligation-induced acute pancreatitis in mice and rats, we found that (a) IL-33 concentration was increased in the pancreas; (b) mast cells, which secrete and also respond to IL-33, showed degranulation in the pancreas and lung; (c) plasma histamine and pancreatic substance P concentrations were increased; and (d) pancreatic and pulmonary proinflammatory cytokine concentrations were increased. In isolated mouse pancreatic acinar cells, TNF-α stimulation increased IL-33 release while IL-33 stimulation increased proinflammatory cytokine release, both involving the ERK MAP kinase pathway; the flavonoid luteolin inhibited IL-33-stimulated IL-6 and CCL2/MCP-1 release. In mice without duct ligation, exogenous IL-33 administration induced pancreatic inflammation without mast cell degranulation or jejunal inflammation; pancreatic changes included multifocal edema and perivascular infiltration by neutrophils and some macrophages. ERK MAP kinase (but not p38 or JNK) and NF-kB subunit p65 were activated in the pancreas of mice receiving exogenous IL-33, and acinar cells isolated from the pancreas of these mice showed increased spontaneous cytokine release (IL-6, CXCL2/MIP-2α). Also, IL-33 activated ERK in human pancreatic tissue. Significance As exogenous IL-33 does not induce jejunal inflammation in the same mice in which it induces pancreatic inflammation, we have discovered a potential role for an IL-33/acinar cell axis in the recruitment of neutrophils and macrophages and the exacerbation of acute pancreatic inflammation. Conclusion IL-33 is induced in acute pancreatitis, activates acinar cell proinflammatory pathways and exacerbates acute pancreatic inflammation.

Dominant Negative p38 Mitogen-Activated Protein Kinase Expression Inhibits NF-κB Activation in AR42J Cells

Background: The role of the p38 mitogen-activated protein (MAP) kinase in acute pancreatitis pathogenesis is controversial. We hypothesize that p38 plays a role in regulating NF-ĸB activation in exocrine pancreatic cells. Methods: AR42J cells incorporating an NF-ĸB-responsive luciferase reporter, with and without adenoviral transduction of DNp38, were stimulated with cholecystokinin (CCK) or tumor necrosis factor-α (TNF-α) prior to measuring NF-ĸB activation. Results: CCK- or TNF-α-stimulated NF-ĸB-dependent gene transcription (luciferase assay) was substantially subdued by DNp38 expression. These findings were confirmed by electrophoretic mobility shift assay. Nuclear translocation of the p65 NF-ĸB subunit following agonist stimulation was evident (supershift). Characterization studies showed excellent adenoviral infection efficiency and cell viability in our AR42J cell model. Agonist-stimulated dose- and time-dependent p38 activation, with inhibition by DNp38 expression, was also confirmed. Conclusion: The p38 MAP kinase regulates NF-ĸB pathway activation in exocrine pancreatic cells, and thus potentially plays a role in the mechanism of acute pancreatitis pathogenesis.

Nuclear factor kappa B-dependent gene transcription in cholecystokinin- and tumor necrosis factor-α-stimulated isolated acinar cells is regulated by p38 mitogen-activated protein kinase

BACKGROUND Mitogen-activated protein (MAP) kinases and nuclear factor kappa B (NF-kappaB) are implicated in early stages of acute pancreatitis pathogenesis. We investigated the relationship between the p38 MAP kinase and NF-kappaB in isolated acinar cells. METHODS Isolated rodent acinar cells were stimulated with agonists after infection with an adenovector containing a luciferase promoter driven only by NF-kappaB and an adenovector containing the dominant negative (DN) form of p38 (empty vector in controls). RESULTS Initial immunoblots confirmed that the agonist stimulated p38 activation in acinar cells was substantially attenuated by DN p38 overexpression. Stimulation of native cholecystokinin (CCK)-A receptors or tumor necrosis factor-alpha (TNF-alpha) receptors promoted a significant increase in NF-kappaB-dependent gene transcription in cells infected with the empty vector, while overexpression of DN p38 significantly abrogated NF-kappaB-dependent luciferase activity. CONCLUSIONS These findings support our hypothesis that p38 is involved in the activation of proinflammatory nuclear transcription factors such as NF-kappaB in pancreatic exocrine cells.

Enteral Exclusion Increases Map Kinase Activation and Cytokine Production in a Model of Gallstone Pancreatitis

Background: We have previously demonstrated that enteral exclusion augments pancreatic p38 mitogen-activated protein (MAP) kinase activation and tumor necrosis factor-α (TNF-α) production after bile-pancreatic duct ligation in rats. Methods: In the present study, we evaluated c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation, and cytokine production, in pancreata of duct-ligated rats with and without duodenal bile-pancreatic juice replacement from a donor rat. We hypothesized that enteral exclusion of bile-pancreatic juice activates stress kinases and induces cytokine production in ligation-induced acute pancreatitis. Results: Increased JNK and ERK activation after ligation are inhibited by bile-pancreatic juice replacement. Increases in pancreatic production of IL-1β and IL-12 after ligation are significantly subdued by replacement. In additional in vitro studies, we show that cholecystokinin- or TNF-α-stimulated nuclear transcription factor kappa-B activation in AR42J cells is inhibited by dominant negative ERK2. Conclusions: Our novel findings using our Donor Rat Model indicate that bile-pancreatic juice exclusion induces MAP kinase activation and exacerbates cell stress and inflammation in this experimental model of gallstone pancreatitis.

Murine pancreatic duct ligation induces stress kinase activation, acute pancreatitis, and acute lung injury

BACKGROUND Acute lung injury is a major determinant of outcomes in acute pancreatitis. We evaluated acute lung injury and stress kinase activation in ligation-induced acute pancreatitis in mice. METHODS Mice with duct ligation or sham operation were killed after 24 or 48 hours. RESULTS In addition to acute pancreatitis, duct ligation was associated with pulmonary morphologic changes indicative of acute lung injury (alveolar septal thickening, congestion, and neutrophil infiltration). Furthermore, immunoblotting showed stress kinase activation in the pancreas and lung after ligation. Although mortality was observed in the ligated group, that is consistent with severe lung injury, it requires further evaluation. CONCLUSIONS Bile and pancreatic duct ligation in the mouse is associated with pancreatic and pulmonary stress kinase activation and acute inflammatory changes consistent with early acute pancreatitis and acute lung injury. Our findings are important as acute lung injury increases mortality in clinical acute pancreatitis and stress kinases are established proinflammatory signal transducers.

A Novel Model of Severe Gallstone Pancreatitis: Murine Pancreatic Duct Ligation Results in Systemic Inflammation and Substantial Mortality

Background: Suitable experimental models of gallstone pancreatitis with systemic inflammation and mortality are limited. We developed a novel murine model of duct-ligation-induced acute pancreatitis associated with multiorgan dysfunction and severe mortality. Methods: Laparotomy was done on C57/BL6 mice followed by pancreatic duct (PD) ligation, bile duct (BD) ligation without PD ligation, or sham operation. Results: Only mice with PD ligation developed acute pancreatitis and had 100% mortality. Pulmonary compliance was significantly reduced after PD ligation but not BD ligation. Bronchoalveolar lavage fluid neutrophil count and interleukin-1β concentration, and the plasma creatinine level, were significantly elevated with PD ligation but not BD ligation. Pancreatic nuclear factor ĸB (p65) and activator protein 1 (c-Jun) were activated within 1 h of PD ligation. Conclusion: PD-ligation-induced acute pancreatitis in mice is associated with systemic inflammation, acute lung injury, multiorgan dysfunction and death. The development of this novel model is an exciting and notable advance in the field.