Oxidized phospholipids exert a dual effect on bile acid-induced CCL2 expression in pancreatic acini.

BACKGROUND: oxidized phospholipids (oxPLs) generated in inflammatory diseases could play a key role by inducing pro- and anti-inflammatory effects. OBJETIVES: we investigated the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and oxidized POPC (oxPOPC) in the inflammatory response triggered in pancreatic acini. METHODS: control acini were incubated in the absence or presence of either POPC or oxPOPC (≤100 µM). In additional experiments, oxPOPC effects were evaluated in sodium taurocholate (NaTc)-treated acini. CCL2 and TLR4 mRNA expression was analyzed by RT-qPCR. By western blot, JNK-MAPK, JAK and IκBα in cytoplasm as well as p65-NF-kB and p-STAT3 in the nucleus were evaluated. The involvement of TLR4, JNK-MAPK, JAK as well as NF-kB, STAT3 and PPARγ was assessed using pharmacological inhibition. RESULTS: no effect was found in response to POPC. Conversely, in response to oxPOPC (10 µM), JNK-MAPK and JAK acted as TLR4-downstream signals, leading to CCL2 upregulation mainly through NF-kB activation. Moreover, TLR4 non-dependent mechanisms induced STAT3 activation in oxPOPC-treated acini. Mediated by PPARγ, oxPOPC (50 µM) inhibited the CCL2 overexpression found in NaTc-treated acini. CONCLUSIONS: oxPOPC exerts pro- and anti-inflammatory effects in pancreatic acinar cells mediated by TLR4 and PPARγ signals, respectively. This dual action proved to be dependent on the concentration. The molecular mechanisms involved in the oxPL response could be useful for new therapeutic approaches to the treatment of oxPLs-related inflammatory pathologies.

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.

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.

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.