Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells

Abstract

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-κB, as assessed by in vitro kinase assays for IκB kinase (IKK), IκBα steady-state levels, p65 nuclear translocation, NF-κB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-κB activation in a similar manner. Both LPS- and lipid A-induced NF-κB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-κB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IκB super repressor (Ad5IκB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-κB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.