The Role Of Lipopolysaccharide On Macrophage-mediated Inflammatory Response In Alcoholic Chronic Pancreatitis

Purpose: Monocytes/macrophages play a critical role in managing innate andadaptive immunity-including inflammatory processes by secretingproinflammatory and chemotactic molecules. Macrophage activation triggeredby TLR4and its ligand lipopolysaccharide (LPS) derived from gram negativebacteria has been proposed as a key player in the pathogenesis of alcoholicliver disease. The aims of this study were to determine the serum levels ofproinflammatory and chemostatic cytokines in patients with chronic alcoholicpancreatitis (CAP) and the effect of LPS on inflammatory cytokine secretion byhuman macrophages, and to evaluate the role of macrophage activation in thepathogenesis of CAP.Methods: The fasting vein blood samples were collected in25normalindividuals and24CAP patients who were assessed within48hours fromsymptom onset. The LPS level in plasma was determined by spectrophatometry.The serum soluble MCP-1(sMCP-1), MIP-1α, Rantes and TGF-β1levels weremeasured by ELISA. Human peripheral blood monocytes were isolated, placedin a six-well plate and cultured in RPMI1640/10%FBS. The adheredmonocytes were maintained in fresh medium for6days in the presence of10ng/ml of GM-CSF. The cells were then cultured in the presence or absence ofalcohol or alcohol plus LPS for another24hours. F4/80and CD14antigenswere detected using immunocytochemistry for analyzing the phenotype ofmonocyte to macrophage transformation. The sMCP-1, MIP-1α, Rantes andTGF-β1levels from supernatants were measured by ELISA.Results: Of24CAP patients,62%of cases had endotoximia (plasma LPSlevels more than2×ULN). The serum sMCP-1, MIP-1α, Rantes and TGF-β1 levels in alcoholic CP patients with endotoximia were higher than those innormal controls and in alcoholic CP patients without endotoximia, and theywere3.7-fold,4.1-fold,1.7-fold,1.3-fold of the alcoholic CP patients withoutendotoximia. In vitro study showed that there were more than98%of6-daytissue culture cells identified as macrophages by F4/80and CD14antigen stains.The supernatant sMCP-1, MIP-1α, Rantes and TGF-β1levels in alcohol plusLPS stimulated-macrophages were significantly higher than those in untreatedcontrol and in alcohol treated alone (p<0.01), and they were1.7-fold,2.8-fold,3.4-fold,1.5-fold of the alcoholic treated group.Discussion: Chronic excess intake of alcohol induces an increase in intestinalpermeability, leading to translocation of gut-derived LPS into pancreas throughblood. Translocated LPS stimulates TLR4on macrophages. Upon activation ofTLR4, macrophages produce cytokines. This study highlights the role of LPSon macrophage-mediated inflammatory response in alcoholic chronicpancreatitis.