Sara Sevillano

Kinetic study of TNF-α production and its regulatory mechanisms in acinar cells during acute pancreatitis induced by bile-pancreatic duct obstruction

Cytokines play a critical role in acute pancreatitis (AP) but the contribution of different cell sources to cytokine production is unclear. Unfortunately, there are no data concerning the molecular mechanisms involved in the inflammatory response in humans during AP. For this reason, the aim of this study was to analyse the ability of acinar cells, in comparison with leukocytes, to produce TNF‐α at different stages of AP induced in rats by bile–pancreatic duct obstruction (BPDO) and to investigate the time course of oxidant‐sensitive mechanisms involved in cytokine production. The role of oxygen free radicals as messengers of the mechanisms underlying acinar cell TNF‐α production was assessed in BPDO rats treated with N‐acetylcysteine (NAC). While monocytes were not able to produce TNF‐α until 12 h after inducing AP, acinar cells triggered TNF‐α production from 6 h after BPDO, at which time the pancreas develops maximal oxidative stress. Phosphorylated p38‐MAPK and activated NF‐κB were detected in acinar cells from 6 h after BPDO. NAC treatment reduced pancreatic glutathione depletion during the early stages of AP and attenuated the activation of p38‐MAPK and NF‐κB for 48 h following BPDO. As a result, acinar cells in NAC‐treated rats failed to produce TNF‐α during AP. In addition, NAC delayed monocyte TNF‐α production, thereby maintaining low TNF‐α levels in plasma during BPDO. In conclusion, acinar cells contribute directly to the inflammatory response during BPDO‐induced AP by producing TNF‐α even before inflammatory cells in the peripheral blood. The blockade of oxidant‐mediated signal transduction pathways induced by NAC treatment prevented acinar cell TNF‐α production. Copyright

Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction.

The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.

Major Pathological Mechanisms of Acute Pancreatitis Are Prevented by N-Acetylcysteine

Aim: To analyze the capability of N-acetylcysteine (NAC) to prevent major intra-acinar pathogenic mechanisms involved in the development of acute pancreatitis (AP). Methods: AP was induced by pancreatic duct obstruction (PDO) in rats. Some animals received NAC (50 mg/kg) 1 h before and 1 h after PDO. During a 24-hour period of PDO, plasma amylase activity and pancreatic glutathione and malondialdehyde levels were measured. Cytosolic Ca2+ levels and enzyme (amylase and trypsinogen) load in acinar cells were also analyzed by flow cytometry, and histological analysis of the pancreas was performed by electron microscopy. Results: NAC avoided glutathione depletion at early AP stages, thereby preventing pancreatic oxidative damage, as reflected by normal malondialdehyde levels. By limiting oxidative stress, NAC treatment effectively prevented the impairment of Ca2+ homeostasis found in acinar cells from early AP onwards, thus protecting the pancreas from damage. In addition, lower quantities of digestive enzymes were accumulated within acinar cells. This finding, together with the significantly lower hyperamylasemia observed in these animals, suggests that NAC treatment palliates the exocytosis blockade induced by PDO. Conclusion: By preventing oxidative stress at early AP stages, NAC administration prevents other pathological mechanisms of AP from being developed inside acinar cells, thus palliating the severity of disease.

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‐α (TNF‐α) production and the contribution of T‐cell‐derived pro‐(TNF‐α) and anti‐(IL‐10) inflammatory mediators. TNF‐α 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‐α 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‐α 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‐α 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.

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.

N‐acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis

Abstract.  Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N‐acetyl‐l‐cysteine (NAC) to prevent the generation of OFR and the changes in the cell‐cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow‐cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF‐α plasma levels were also measured. The distribution of acinar cells throughout the different cell‐cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO‐induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper‐amylasaemia and maintenance of plasma TNF‐α levels at values not significantly different from controls were found. NAC administration inhibited progression of cell‐cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S‐phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO‐induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell‐cycle phases. Prevention of OFR generation of acinar cells in rats with PDO‐induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.

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.

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.