The Role of Lipopolysaccharide on Macrophage-Mediated Inflammatory Response in Alcoholic Chronic Pancreatitis

Abstract

Introduction: Monocytes/macrophages play a critical role in managing innate and adaptive immunity-including inflammatory processes by secreting proinflammatory molecules. Macrophage activation triggered by TLR4 and its ligand lipopolysaccharide (LPS) derived from gram-negative bacteria has been proposed as a key player in the pathogenesis of alcoholic liver disease. The aims of this study were to determine the serum levels of proinflammatory cytokines in patients with alcoholic chronic pancreatitis (CP) and the effect of LPS on inflammatory cytokine secretion by human macrophages, and to evaluate the role of macrophage activation in the pathogenesis of alcoholic CP. Methods: The fasting vein blood samples were collected in 30 normal individuals and 45 alcoholic CP patients who were assessed within 48 hours from symptom onset. The LPS level in plasma was determined by spectrophatometry. The serum soluble MCP-1(sMCP-1), MIP-1α, RANTES and TGFβ1 levels were measured by ELISA. Human peripheral blood monocytes were isolated, placed in a 6-well plate and cultured in RPMI 1640/10 % FBS. The adhered monocytes were maintained in fresh medium for 6 days in the presence of 10 ng/ml of GM-CSF. The cells were then cultured in the presence or absence of alcohol or alcohol plus LPS for another 24 hours. F4/80 and CD14 antigens were detected using immunocytochemistry for analyzing the phenotype of monocyte to macrophage transformation. The sMCP-1, MIP-1α, Rantes and TGF-β1 levels from supernatants were measured by ELISA. Results: Of 45 alcoholic CP patients, 60% of cases had endotoximia (plasma LPS levels more than 2×ULN). The serum sMCP-1, MIP-1α, RANTES and TGF-β1 levels in alcoholic CP patients with endotoximia were higher than those in normal controls and in alcoholic CP patients without endotoximia, and they were 3.7-fold, 4.1-fold, 1.7-fold, 1.3-fold of the alcoholic CP patients without endotoximia. In vitro study showed that there were more than 98% of 6-day tissue culture cells identified as macrophages by F4/80 and CD14 antigen stains. The supernatant sMCP-1, MIP-1α, Rantes and TGF-β1 levels in alcohol plus LPS stimulated-macrophages were significantly higher than those in untreated control and in alcohol treated alone (p<0.01), and they were 1.7-fold, 2.8-fold, 3.4-fold, 1.5-fold of the alcoholic treated group. Conclusion: Chronic excess intake of alcohol induces an increase in intestinal permeability, leading to translocation of gut-derived LPS into pancreas through blood. Translocated LPS stimulates TLR4 on macrophages. Upon activation of TLR4, macrophages produce cytokines. This study highlights the role of LPS on macrophage-mediated inflammatory response in alcoholic chronic pancreatitis.