Isabel De Dios

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

Biliary pancreatitis-associated ascitic fluid activates the production of tumor necrosis factor-α in acinar cells*

Objective:Acute pancreatitis is associated with increased cytokine release from different cell sources. We have investigated the ability of acinar cells, in comparison with inflammatory peripheral blood cells, to produce tumor necrosis factor (TNF)-&agr; in response to pancreatitis-associated ascitic fluid (PAAF). Design:Controlled, randomized animal study. Setting:University research laboratory. Subjects:Male Wistar rats. Interventions:Flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-&agr; antiserum. Measurements and Main Results:PAAF (20%, v:v) obtained from rats with acute pancreatitis induced by bile-pancreatic duct obstruction significantly increased TNF-&agr; production in acinar cells, as measured by flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-&agr; antiserum. Neither heating of PAAF nor the addition of soybean trypsin inhibitor or neutralizing amounts of anti-TNF-&agr; monoclonal antiserum reduced the acinar cell TNF-&agr; production. Monocytes and lymphocytes did not produce TNF-&agr; in response to PAAF. Likewise, the typical monocyte and lymphocyte stimulating factors—lipopolysaccharide (10 &mgr;g/&mgr;L) and phorbol 12-myristate 13-acetate (250 ng/mL) plus ionomycin (1 &mgr;g/mL), respectively—were not able to produce TNF-&agr; in acinar cells. By comparison of the two acinar cell populations differentiated by flow cytometry, R2 cells (with higher forward scatter values) showed a greater ability to produce TNF-&agr; in response to PAAF than R1 cells. Acinar cell nuclear factor-&kgr;B was activated, but TNF-&agr; production was not totally inhibited in presence of N-acetyl cysteine (30, 100 mM). Conclusions:The production of TNF-&agr; from different cell sources is selectively activated. PAAF may be involved in the pathophysiology of acute pancreatitis by TNF-&agr; production in acinar cells through mechanisms partially mediated by nuclear factor-&kgr;B activation. PAAF components, such as TNF-&agr; or trypsin, are not responsible for acinar cell activation. TNF-&agr; was induced by heat-resistant PAAF factors, displaying acinar cells with higher forward scatter (R2) a greater ability to increase the TNF-&agr; production than R1 cells.

Signal transduction of MCP-1 expression induced by pancreatitis-associated ascitic fluid in pancreatic acinar cells

Pancreatitis‐associated ascitic fluid (PAAF) is known to contribute to the progression of acute pancreatitis (AP). We have investigated the capability of PAAF to activate the expression of MCP‐1 in pancreatic acinar cells and the involvement of MAPK, NF‐κB and STAT3 as downstream signalling transduction pathways. The actions of dexamethasone (Dx) and N‐acetylcysteine (NAC) on the PAAFs acinar effects have also been evaluated. Acinar cells were incubated for 1 hr with PAAF collected from rats with severe AP induced by sodium taurocholate in the absence or presence of Dx (10−7 M) or NAC (30 mM). MCP‐1 mRNA expression, phospho‐p38‐MAPK, IκBα, nuclear p65 levels and nuclear translocation of STAT3 were analysed. In response to PAAF, overexpression of MCP‐1, phosphorylation of p38‐MAPK, degradation of IκBα and increases in p65 nuclear levels and STAT3 activity were found in acinar cells. PAAF‐mediated MCP‐1 up‐regulation was completely suppressed by Dx and NAC. MAPK activation was only inhibited by NAC, NF‐κB activation was repressed by Dx and NAC, and STAT3 pathway was strongly blocked by Dx and significantly reduced by NAC. In conclusion, acinar cells were activated by PAAF to produce MCP‐1, mainly via NF‐κB and STAT3 pathways. Both downstream pathways were targeted by Dx and NAC to repress the PAAF‐mediated acinar MCP‐1 up‐regulation.

Cerulein-induced acute pancreatitis in the rat : study of pancreatic secretion and plasma VIP and secretin levels

A study was made with different doses of cerulein (2, 4, 10 and 20 μg/kg) administered subcutaneously to rats by four injections at intervals of 1 hr; the aim of this work was to study exocrine pancreatic secretion of the rat under cerulein-induced acute pancreatitis, analyzing enzyme and hydroelectrolyte secretion of pancreatic juice. A further aim was to study the relationship between the dose of cerulein and the plasma levels of peptides controlling hydroelectrolyte secretion of the pancreas, like secretin and vasoactive intestinal peptide (VIP). At the lowest dose schedule, the amounts of total protein and enzymes (amylase and trypsin) in pancreatic juice decreased significantly, plasma amylase increased, and the pancreas became edematous. Higher doses magnified these effects. By contrast, ductular function (flow and HCO3−) was well preserved in ceruleintreated rats, and this was probably due to the significant increase in plasma levels of immunoreactive secretin whereas VIP levels were unchanged. The secretin released by treatment with cerulein is able to palliate the lack of flow from acinar origin that is affected in the process of acute pancreatitis, being a beneficial response to the cerulein treatment.

CD45 expression on rat acinar cells: involvement in pro-inflammatory cytokine production.

CD45 transduces activation signals in inflammatory cells. We investigate CD45 expression on pancreatic acinar cells and examine its role in the inflammatory response which these cells have also shown under certain circumstances. Similar CD45 mRNA levels were found in acinar cells and leukocytes (positive control). Flowcytometric and immunohistochemical analysis showed a heterogeneous CD45 distribution on acinar cells. Activation of acinar cells by incubation with pancreatitis‐associated ascitic fluid as evidencied by TNF‐〈alpha〉 production resulted in a decreased CD45 expression, suggesting that CD45 acts as a negative regulator of cytokine production. As a validation of this finding in vivo, a decrease in the acinar CD45 expression in parallel with an increased ability to produce TNF‐〈alpha〉 was found in rats with acute pancreatitis. Our data show that CD45 is constitutively expressed in acinar cells and suggest that it plays an important role in negatively regulating cytokine production.

Enzyme Changes in Zymogen Granules and in Pancreatic Secretion Throughout Long-Term CCK Treatment

Pancreatic enzyme storage and secretion were studied in rats treated twice daily with s.c. injections (5 micrograms/kg) of CCK-8 for 3, 7, and 15 days. Isolated zymogen granules were analyzed by flow cytometry to determine their FSC (forward scatter), SSC (side scatter), and amylase and trypsinogen contents. DNA content, pancreatic weight, and both basal and stimulated pancreatic secretion under i.v. CCK infusion (1.25 micrograms/kg/h) were also studies. Two subsets of zymogen granules were identified by flow cytometry in both control and CCK-treated rats on the basis of FSC and SSC parameters: Z1 (smaller and less complex) and Z2. Both subsets displayed a high degree of heterogeneity with respect to their enzyme content per zymogen granule. During the first 7 days of CCK treatment, hyperplasia and hypertrophy developed in the rats together with changes in the zymogen granules, reflected by a significantly decreased FSC, and increased SSC, and an increase in the mean trypsinogen/amylase ratio per granule. A rise in pancreatic enzyme secretion, especially of trypsin, was observed. After 15 days of CCK administration, a simultaneous decrease in amylase content and increase in trypsinogen content per zymogen granule was observed. A desensitization of the pancreas to CCK happened after 15 days of CCK administration, reflected by a reduction of all the pancreatic functions that had been increased at shorter CCK administration periods. Nevertheless, trypsinogen appeared resistant to desensitization because its secretion significantly increased in response to an i.v. infusion of CCK. CCK treatment displayed a differential packaging of the enzymes in individual zymogen granules; the trypsinogen/amylase ration was significantly higher in Z2 zymogen granules than in Z1 subset throughout the treatment.

Targeting peripheral immune response reduces the severity of necrotizing acute pancreatitis.

Objective:A complex cascade of immunologic events leads to the development of systemic inflammatory response in acute pancreatitis (AP). Our aim was to evaluate the effects of two different immunomodulating treatments: Dexamethasone (Dx) and N-acetylcysteine (NAC), on the progression of necrotizing AP. Design:Prospective, random, and control study. Laboratory animals. Setting:University-based research laboratory. Subjects:Male Wistar rats. Interventions:Retrograde infusion of 3.5% of sodium taurocholate into pancreatic-biliary duct was used to induce AP in rats. Dx (1 mg/kg) was administered 30 mins before or 1 hr after AP, and NAC (50 mg/kg) was given 1 hr before and 1 hr after inducing AP. Measurements and Main Results:Dx and NAC treatments reduced the severity of AP in terms of amylasemia, pancreatic edema, and pancreatic and liver necrosis. Dx, administered before or after AP, and NAC reduced the leukocytosis induced by AP and blocked the ability of circulating monocytes to produce tumor necrosis factor-α and monocyte chemoattractant protein-1; however none of them significantly reduced the overexpression of intercellular cell adhesion molecule-1 found in monocytes 6 hrs after inducing AP. Leukocyte infiltration in the pancreas was attenuated in Dx-pretreated rats and significantly reduced 6 hrs after inducing AP in rats treated with NAC. However, neither Dx nor NAC were able to significantly reduce interleukin-6 in plasma or mitigate leukocyte infiltration in the lung. Conclusions:Our data demonstrated that treatments targeting the peripheral immune response reduced the severity of sodium taurocholate -induced AP attenuating pancreatic and liver injury, but they were not effective for limiting the spread of the inflammatory damage to the lung.

Extrapancreatic organ impairment during acute pancreatitis induced by bile-pancreatic duct obstruction. Effect of N-acetylcysteine

Multiple organ failure is frequently associated with acute pancreatitis (AP). Our aim was to study pulmonary, hepatic and renal complications developed in the course of AP experimentally induced in rats by bile‐pancreatic duct obstruction (BPDO), differentiating the complications caused by AP itself, from those directly caused by bile duct obstruction (BDO), after ligating the choledocus. N‐acetylcysteine (NAC) was administered as a therapeutic approach. Myeloperoxidase activity revealed neutrophil infiltration in lungs from 12 h after BDO, even if AP was not triggered. Lactate dehydrogenase (LDH) activity indicated hepatocyte death from 48 h after BDO, and from 24 h following BPDO‐induced AP onwards, an effect delayed until 48 h by NAC treatment. Rats with single cholestasis (BDO) and rats with BPDO‐induced AP showed a significant increase in plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin concentration from 12 h onwards, whose values were reduced by NAC treatment at early BPDO. No renal failure was found during 120 h of bile‐pancreatic obstruction. Our results showed lung and liver impairment as a result of BDO, even if AP does not develop. Pancreatic damage and extrapancreatic complications during AP induced by BPDO were palliated by NAC treatment.

Enzyme load in pancreatic acinar cells is increased in the early stages of acute pancreatitis induced by duct obstruction in rats

Trypsinogen and amylase content has been analysed by flow cytometry in individual pancreatic cells from rats with acute pancreatitis induced by pancreatic duct obstruction, from the earliest stages to 48 h after obstruction. Parallel morphological studies of the pancreas by electron microscopy and analysis of various parameters for the diagnosis of pancreatitis will allow research into the possible relationship between intracellular enzyme load and the severity of pancreatitis. Progressive increases in amylase activity in ascites and plasma, the volume of ascites, haematocrit, vacuolization, oedema and macrophage infiltration were observed between 1.5 h and 12 h after duct obstruction. A progressive increase in enzyme content was also observed in individual acinar cells at this stage. Interestingly, the larger increase was for trypsinogen, so that the trypsinogen/amylase ratio was significantly increased in all acinar cells by 12 h after duct obstruction. This represents a risk factor for the development of pancreatitis. Sections of pancreas taken from rats that had duct obstruction for 48 h showed massive dilatation and disorganization of the endoplasmic reticulum, focal apoptosis and necrosis. These severe alterations would affect enzyme synthesis, as reflected by the significant decrease in the intracellular enzyme load observed at this stage. However, not all acinar cells were affected equally by the damage induced by pancreatitis: R(1) cells appeared to be more sensitive than R(2) cells. In conclusion, intracellular accumulation of digestive enzymes occurs at early stages of pancreatitis, and this effect is proportionally greater for trypsinogen, a finding that could explain the degree of severity achieved in the course of pancreatitis.

Exocrine pancreatic response to CCK in rats after long-term hydrocortisone administration

The effects of prolonged administration of hydrocortisone (10 mg/kg/day) on exocrine pancreatic secretion were examined, analyzing the quantitative and qualitative variations in secretion at 7, 18 and 30 days of treatment. The weight of the pancreata was found to increase during the period of hydrocortisone treatment. After the 7th day of treatment the hormone significantly decreases basal exocrine pancreatic secretion, maintaining similar values up to the 30th day of treatment. Hydrocortisone enhances the pancreatic response to CCK since the percent increase in acinar secretion under stimulation compared to basal secretion surpassed control levels in all cases. However, the inhibitory effect on secretion shown by hydrocortisone opposes and surpasses the stimulatory effect of the secretagogue, secretion being reduced in the treated animals. Hydrocortisone especially affected the amylase fraction of the juice, to a larger extent inhibiting the isoenzyme A1, with an IEP of 8.5 when the animals were treated over 30 days; thus, a considerable reduction was observed in the amylase secreted both in resting conditions and under stimulation with CCK. There is a possibility that chronic treatment with glucocorticoids may sensitize the acinar cells, alter the composition of the pancreatic juice and inhibit secretion, effects that may involve pancreatic dysfunction.