THE INFLAMMATORY RESPONSE OF THE PANCREATIC ACINAR CELL

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DESCRIPTION (provided by applicant): The pancreatic acinar cell has served as a cell biologic model for determining the mechanisms of protein synthesis, transport and secretion regulated by neurohumoral agents. In contrast, little is known about the pancreatic acinar cell in pathologic conditions such as pancreatitis and pancreatic cancer. We have preliminary data showing that the key agonist for pancreatic acinar cells, the hormone cholecystokinin (CCK), causes activation of intracellular signals (i.e. transcription factors and kinases) that regulate the production of proinflammatory cytokines and chemokines. We also have preliminary data showing that the pancreatic acinar cell responds to cytokines such as tumor necrosis factor (TNF-alpha) and that this agent modifies the effect of CCK on the intracellular signals in the pancreatic acinar cell that mediate the generation of the inflammatory molecules (cytokines, adhesion molecules, etc.). For the present application, we hypothesize that in the pancreatic acinar cell CCK activates intracellular signals modulated by TNF-alpha that mediate the expression of the inflammatory molecules and pancreatitis. The specific objectives for the present application are: (1). Determine the effects of CCK and TNF-alpha alone and in combination on activation and composition of the transcription factors, NF-kappa-B and AP-1, in pancreatic acinar cells in vitro. (2). Determine the intracellular signaling systems involved in mediating the effects of CCK and TNF-alpha on transcription factor activation. (3). Determine the effect of TNF-alpha on CCK-induced expression of cytokines in pancreatic acinar cells. (4). Determine the roles of specific NF-kappa-B proteins (i.e. p65, p50 and c-Rel) in regulating cytokine expression in pancreatic acinar cells in vitro. (5). Determine the roles of the specific NF-kappa-B proteins in regulating pancreatic cytokine expression and the inflammatory and cell death responses of experimental pancreatitis. In vitro experiments in this application will use acinar cells prepared by collagenase digestion of pancreas from rat or mouse. In vivo experiments will use mice with genetic deletions of various NF-kappa-B proteins as well as wild-type controls. Cytokines will be assayed using RT-PCR, Northern and Western blot analyses, and immunocytochemistry. Intracellular signals measured will include transcription factor activation, kinase activities, and second messengers measured with gel shift assay, Western blot analysis, enzyme assays, radioimmunoassay, and intracellular Ca2+ concentrations. Measures of pancreatitis will include both morphologic (including immunohistochemical) techniques and biochemical determinations of transcription factor activation, cytokine expression, and trypsin activation in the pancreatic tissue. The results of the experiments described in the present application will allow us to propose how CCK, together with a cytokine modulating its effects, acts on the pancreatic acinar cell to cause experimental pancreatitis.