Cholecystokinin blockade alters the systemic immune response in rats with acute pancreatitis.

Acute pancreatitis (AP) is characterized by initial pancreatic injury resulting from the activation of digestive enzymes and, later, widespread inflammation to distant organs. The aim of this study was to study whether the time-course of inflammatory events during AP induced by bile-pancreatic duct obstruction (BPDO) varies after lowering the acinar enzyme content by L364,718 (0.1 mg/kg/day) administration over 7 days before inducing AP. Flow cytometric immunophenotyping was used to analyse the following at different AP stages: distribution of major circulating leucocyte subsets, activation state of circulating neutrophils and monocytes as reflected by CD11b expression and tumour necrosis factor-alpha (TNF-alpha) production and the contribution of T-cell-derived pro-(TNF-alpha) and anti-(IL-10) inflammatory mediators. TNF-alpha plasma levels and neutrophil infiltration in pancreas and lung were also measured. At early BPDO times, L364,718 treatment partially inhibited leukocytosis and increase in peripheral blood neutrophils and monocytes as well as TNF-alpha expression by monocytes. However, from 6 h onwards after BPDO, L364,718 treatment was unable to prevent either pancreatic and lung neutrophil infiltration or the release of TNF-alpha from activated monocytes. By its action on circulating lymphocytes, L364,718 treatment enhanced the severity of the inflammatory response induced by BPDO. Peripheral blood lymphocytes were recruited from earlier BPDO times, and 12 h after BPDO, T cells displayed a significantly higher reserve of TNF-alpha able to be released under stimulation but lower functional reserve of interleukin-10 (IL-10) than observed in untreated rats. It is concluded that lowering the acinar enzyme content through L364,718 treatment prevents earlier systemic immune events in BPDO-induced AP. However, at the point of maximal injury, the inflammatory response became pronounced, largely due to the role played by activated T lymphocytes.

Effect of long-term CCK blockade on the pancreatic acinar cell renewal in rats with acute pancreatitis.

This study determines the effect of 7-day pretreatment with L364,718 (a potent cholecystokinin (CCK) receptor antagonist) on pancreatic cell turnover during the course of acute pancreatitis (AP) induced in the rat by bile-pancreatic duct obstruction (BPDO). Cell cycle distribution and apoptosis were analyzed by flow cytometry using propidium iodide (PI) and Annexin V staining. Besides altering the pancreatic redox status, long-term CCK blockade inhibited the normal proliferation of acinar cells as indicated by the significant increase in G(0)/G(1)-phase cells and the decrease in G(2)/M-cells found in control rats treated with L364,718 for 7 days. A progressive depletion in pancreatic GSH was found from 3 to 24h after BPDO with similar values in L364,718-pretreated and non-treated rats, which led to a maximum peak in malondialdehyde (MDA) levels 6h after BPDO. However, plasma amylase activity and ascites volume indicated higher severity of AP in L364,718-pretreated rats. CCK blockade enhanced the alterations that appear in cell cycle distribution of acinar cells during AP demonstrated by the significantly higher increase in G(0)/G(1)-cells and decrease in S-cells found in L364,718-treated rats 48h after BPDO. Our results indicate that the renewal of acinar cells deleted by apoptosis 48h after BPDO worsens if CCK is blocked before inducing AP.

Cholecystokinin blockade triggers earlier and enhanced intra-acinar oxygen free radical generation in acute pancreatitis induced by pancreatic duct obstruction in rats.

Cholecystokinin (CCK) has been suggested to be a contributory mediator in acute pancreatitis (AP). The aim of the present study was to assess the role of CCK in the development of oxidative stress at different stages of AP induced by pancreatic duct obstruction (PDO) in rats, using L364,718 (a potent CCK-receptor antagonist) to block CCK action. Intra-acinar oxygen free radical (OFR) generation was analysed by flow cytometry using dihydrorhodamine-123 as a fluorogenic dye. Parallel measurements of pancreatic levels of reduced glutathione (GSH) and of several parameters for the diagnosis of AP were performed in both untreated PDO rats and PDO rats receiving L364,718 (0.1 mg x 12 h(-1) x kg(-1)). Diagnosis parameters indicated a greater severity of AP in rats treated with the CCK antagonist. The increase in OFR generation observed in acinar cells up to 12 h after inducing AP was triggered at an earlier stages and reached higher values when L364,718 was administered. Accordingly, greater pancreatic GSH depletion was observed in rats with AP treated with the CCK antagonist. Two populations of acinar cells that were differentiated by flow cytometry, R1 and R2, showed similar behaviour with regard to OFR generation in PDO rats; however, R1 cells showed greater sensitivity to L364,718 administration, and thus OFR production was increased in R1 cells earlier than in R2 cells. In conclusion, CCK blockade anticipates and enhances the amount of OFR produced in acinar cells as a consequence of AP, thus leading to earlier development of and more severe disease. The detrimental effect of L364,718 in AP induced by PDO suggests that plasma CCK does not play a major role in the development of this AP model.

Low enzyme content in the pancreas does not reduce the severity of acute pancreatitis induced by bile-pancreatic duct obstruction.

Enzyme load in pancreas has been considered a risk factor in the development of acute pancreatitis. In order to confirm this hypothesis our aim was to analyze the development and evolution of acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) after reducing the pancreatic enzyme content. L-364,718 - a potent CCK-receptor antagonist - was administered (0.1 mg/kg/day) for 7 days before inducing AP by BPDO. The course of AP was evaluated at different times from 1.5-48 h after BPDO. Amylase and trypsinogen contents and cytosolic calcium levels were measured by flow cytometry using specific antisera against pancreatic enzymes labelled with isothiocyanate of fluorescein and Fluo 3, respectively. The severity of the disease at the different stages was evaluated by measurements of amylase activity in ascites and plasma, percentage of pancreatic fluid and haematocrit. Electron microscopy study of the pancreas showed an increased number of zymogen granules spread through the acinar cells of control rats treated with L-364,718 for 7 days, however, total enzyme content in individual acinar cells was significantly (p < 0.01) diminished. AP significantly increased intracellular amylase and trypsinogen load from 3-12 h after BPDO, and prior L-364,718 treatment enhanced the blockade of enzyme secretion. As a result, acinar enzyme content was significantly increased from earlier stages (1.5 h after BPDO). In parallel, increased cytosolic calcium levels observed up to 24 h after BPDO appeared earlier in L-364,718-treated rats than in those not treated. The severity of AP seems to have been higher in rats previously treated with the CCK-receptor antagonist as indicated by the significantly higher pancreatic fluid and amylase activity in ascites and plasma observed at different times after BPDO. Our results indicate that there is no correlation between the severity of pancreatitis and the amount of enzymes accumulated in the pancreas before the disease is induced.

Asynchronous impairment of calcium homoeostasis in different acinar cells after pancreatic duct obstruction in rat.

Current evidence suggests that alterations within acinar cells are responsible for the development of acute pancreatitis. After inducing acute pancreatitis in rats by pancreatic duct obstruction, we analysed, using flow cytometry, the progressive changes in cytosolic Ca2+ concentrations in individual acinar cells from the earliest stages to 48 h after obstruction to investigate whether parallel alterations in the homoeostasis of Ca2+ could be defined in the different acinar cells throughout the evolution of pancreatitis. Morphological alterations of the pancreas, related to the severity of the disease at different stages, were observed by electron microscopy. Hyperamylasaemia and progressively more severe alterations, such as vacuolization, dilatation of endoplasmic reticulum, accumulation of zymogen granules and reorientation towards basolateral membrane, were observed during the first 12 h after pancreatic obstruction. A significant increase in cytosolic Ca2+ concentration was measured at these stages in a particular type of acinar cells (R1) differentiated by flow cytometry with low forward scatter (FSC), whereas another representative group of cells (R2) with higher FSC values were able to maintain resting cytosolic Ca2+ concentrations up to 24 h after obstruction. Longer periods of pancreatic duct obstruction induced disturbances in Ca2+ homoeostasis in all acinar cells. A similar increase in cytosolic Ca2+ load was reached in both R1 and R2 cells when acute pancreatitis was completely developed. In conclusion, the homoeostasis of Ca2+ in acinar cells is asynchronously impaired during the development of acute pancreatitis; cells with higher FSC (R2) appear to be more resistant than R1 cells.

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats.

Little information is available regarding the role of circulating leukocytes in the pathogenesis of acute pancreatitis (AP). Our aim was to explore the time-course of the potential role of inflammatory peripheral blood (PB) cells during AP induced in rats by pancreatic duct obstruction (PDO). Flow cytometry immunophenotyping was used to analyse the distribution of the major circulating leukocyte subsets, the activation state of circulating monocytes as reflected by both CD11b expression and TNF-alpha production and the relative contribution of T-cell derived pro- (TNF-alpha) and anti- (IL-10) inflammatory mediators at different stages of PDO-induced AP. A progressive increase in PB neutrophils and monocytes was observed up to 6h after PDO whereas lymphocytes, as well as CD4(+) and CD8(+) T-cell subsets, rose as early as 1.5 h after PDO and decreased thereafter. Monocytes were activated in PB from 6 h after inducing AP as reflected by increases in both CD11b expression and spontaneous TNF-alpha production; nevertheless, they showed the capability of producing TNF-alpha at earlier AP stages by lipopolysaccharide (LPS) stimulation. In contrast, T-cells were unable to produce TNF-alpha during AP neither spontaneously nor after stimulation with PMA/Ionomycin. Therefore, only PB monocytes contribute to increase TNF-alpha levels in plasma as observed from 12 h onwards after inducing AP. Interleukin-10 was produced by T-cells 6 h after PDO only after PMA/Ionomycin stimulation. We conclude that systemic inflammatory events are triggered off at early stages of PDO-induced AP, with the activation of circulating monocytes, though not T-cells, playing a central role.