Manuel A. Manso

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.

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.

Mechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pancreatitis

This study aimed to investigate the role of therapeutic dexamethasone (Dex) treatment on the mechanisms underlying chemokine expression during mild and severe acute pancreatitis (AP) experimentally induced in rats. Regardless of the AP severity, Dex (1 mg/kg), administered 1 h after AP, reduced the acinar cell activation of extracellular signal-regulated kinase (ERK) and c-Jun-NH(2)-terminal kinase (JNK) but failed to reduce p38-mitogen-activated protein kinase (MAPK) in severe AP. In both AP models, Dex inhibited the activation of nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT) factors. All of this resulted in pancreatic down-regulation of the chemokines monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC). Lower plasma chemokine levels as well as decreased amylasemia, hematocrit and plasma interleukin-1beta (Il-1beta) levels were found either in mild or severe AP treated with Dex. Pancreatic neutrophil infiltration was attenuated by Dex in mild but not in severe AP. In conclusion, by targeting MAPKs, NF-kappaB and STAT3 pathways, Dex treatment down-regulated the chemokine expression in different cell sources during mild and severe AP, resulting in decreased severity of the disease.

Effect of chronic administration of hydrocortisone on the induction and evolution of acute pancreatitis induced by cerulein

The effects of chronic administration of hydrocortisone (10 mg/kg/day) on the development and evolution of acute pancreatitis induced by supramaximal stimulation with cerulein were examined in the rat. In these circumstances the potentially therapeutic effect of L-364,718, a CCK-receptor antagonist, was assayed. Administration of hydrocortisone over 7 days did not increase the severity of edematous acute pancreatitis induced by cerulein, since the reduction in pancreatic secretion, the hyperamylasemia and the increase in the levels of hematocrit and fluid in the pancreatic tissue were similar in rats with acute pancreatitis treated and untreated with hydrocortisone previously. When hydrocortisone was administered chronically, before administration of supramaximal doses of cerulein, a spontaneous regression of acute pancreatitis occurred. However, when hydrocortisone administration was continued after inducing pancreatitis, pancreatic recovery was prevented, observing a significantly depressed acinar secretion and elevated values of hematocrit and tissue fluid (edema). L-364,718 administration proved to be detrimental in the evolution of edematous acute pancreatitis when the rats had been treated chronically with hydrocortisone because the blockade exerted on secretion prevented the draining of enzymes stored in excess by hydrocortisone administration.

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.

Therapeutic and protective effect of subcutaneous injections of L-364,718 on caerulein-induced acute pancreatitis.

The prophylactic and therapeutic effects of a potent cholecystokinin (CCK) receptor antagonist, L-364,718, on acute pancreatitis induced by caerulein were evaluated, analyzing morphologic and functional pancreatic parameters jointly. Edematous pancreatitis was induced by four subcutaneous injections of caerulein (20 μkg) in rats at 1-h intervals. Prophylactic administration of L-364,718 (0.1 mg/kg) prevented rise in serum amylase levels, interstitial edema, vacuolization, and impairment of pancreatic enzyme secretion that accompany caerulein-induced acute pancreatitis. After 7 days, a spontaneous regression of the morphologic alterations caused by caerulein-induced acute pancreatitis occurs; however, recovery of the secretory function of the pancreas was only reached after this period of time when L-364,718 was administered therapeutically (0.1 mg/kg/ day). Prophylactically or therapeutically administered, L-364,718 exerts a beneficial effect on caerulein-induced acute pancreatitis, indicating that CCK (exogenous or endogenous) plays an important role in the development of this pathology.