Laura Ramudo

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.

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.

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.

The role of redox status on chemokine expression in acute pancreatitis.

This study focused on the involvement of oxidative stress in the mechanisms mediating chemokine production in different cell sources during mild and severe acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) and 3.5% NaTc, respectively. N-Acetylcysteine (NAC) was used as antioxidant treatment. Pancreatic glutathione depletion, acinar overexpression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC), and activation of p38MAPK, NF-kappaB and STAT3 were found in both AP models. NAC reduced the depletion of glutathione in BPDO- but not in NaTc-induced AP, in which oxidative stress overwhelmed the antioxidant capability of NAC. As a result, inhibition of the acinar chemokine expression and signalling pathways occurs in mild, but not in severe AP. However, MCP-1 and CINC expressions in whole pancreas and plasma chemokine levels were not reduced by NAC, even in BPDO-induced AP, suggesting that in addition to acini, other pancreatic cells produced chemokines by antioxidant resistant mechanisms. The high Il-6 plasma levels found during AP, both in NAC-treated and non-treated rats, pointed out cytokines as activating factors of chemokine expression in non-acinar cells. In conclusion, from early AP oxidant-mediated MAPK, NF-kappaB and STAT3 activation triggers the chemokine expression in acini but not in non-acinar cells.

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.

Redox-sensitive modulation of CD45 expression in pancreatic acinar cells during acute pancreatitis.

CD45, a transmembrane protein tyrosine phosphatase required for signal transduction in leukocytes, has recently been found in pancreatic acinar cells. We have investigated the relationship between kinetic expression of CD45 on acinar cells during acute pancreatitis (AP) and the ability of these cells to produce tumour necrosis factor‐α (TNF‐α) through mechanisms sensitive to the cellular redox state. Flow cytometric analysis showed a significant decrease in the constitutive expression of CD45 in acinar cells from six hours onwards after inducing AP by bile‐pancreatic duct obstruction (BPDO) in parallel with a significant increase in acinar TNF‐α production. Changes in protein expression on the acinar cell surface preceded CD45 mRNA down‐regulation, which was not found until 12 hours after BPDO. N‐Acetylcysteine treatment delayed and reduced the down‐regulation of CD45 expression induced by AP and prevented acinar cells from producing TNF‐α. Our results show that CD45 expression is down‐regulated in acinar cells during acute pancreatitis by redox‐sensitive mechanisms, and they support the notion that CD45 negatively controls the production of cytokines in pancreatic acinar cells. Copyright

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.