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Intracellular Ca2؉ -changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [ influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca 2؉ -signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.Alterations in intracellular calcium signaling have previously been reported from an experimental animal model of acute pancreatitis that employs supramaximal secretagogue stimulation for the disease induction (1). Moreover, the spatial and temporal distribution of intracellular calcium signals in response to either physiological or pathological stimuli has been found to be directly related to premature digestive enzyme activation in the pancreas and to acinar cell injury (2-5). Intracellular activation of trypsinogen on the other hand can be completely prevented with agents that interfere with either the uptake of calcium, the maintenance of a calcium gradient across the plasma membrane, or the rapid release of calcium from apical intracellular stores (4, 6). All of these studies indicate clearly that the spatial and temporal distribution of intracellular calcium signals plays a critical role in the early cellular events that precede the onset of pancreatitis. The mechanism, however, that was used to induce acinar cell injury in these studies or in response to which intracellular calcium changes were characterized, supramaximal secretagogue stimulation, is not necessarily an event that is generally involved in the pathogenesis of clinical pancreatitis.In many parts of the world the most common etiological factor associated with acute pancreatitis is gallstone disease. Experimental (7) as well as clinical studies (8, 9) suggest that the onset of gallstone-induced pancreatitis requires migration of the offending stone through the biliary tract and its impaction at the duodenal papilla. Here, at the junction of the common bile duct and the pancreatic duct, the stone can impair the flow of pancreatic secretion or lead to a complete blockage of the pancreatic duct. It is now generally accepted that this impairment of pancreatic secretion (7, 10, 11), rather than a potential reflux of bile into the pancreas (13), represents the critical pathophysiological event for the development gallstoneinduced pancreatitis. To investigate whether this clinically relevant...

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

SUMMARYIntestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll±Hypaque gradient followed by coincubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-