Normal rat hepatic stellate cells respond to endotoxin in LBP‐independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

Abstract

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.

Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: Summary of a symposium

“…Activated mouse HSCs expressed TLR-4 and CD-14 and responded to LPS with an up regulation of extracellular signal-regulated kinase phosphorylation and IL-6, transforming growth factor-β1, and MCP-1 secretion (Brun et al, 2005). In quiescent rat hepatic stellate cells, LPS stimulated the synthesis of proinflammatory cytokines TNF-α, IL-6, and IL-1 (Thirunavukkarasu et al, 2005). These results suggest that LPS may directly activate HSCs, thereby contributing to liver fibrosis.…”

Mechanisms of endotoxin-induced NO, IL-6, and TNF-α production in activated rat hepatic stellate cells: Role of p38 MAPK

“…The protocols were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh in accordance with National Institutes of Health regulations. HSCs were isolated from male Sprague-Dawley rats (450-500 g) as previously described 21,22 via collagenase and protease digestion of the liver, followed by removal of hepatocytes and cell debris via low-speed centrifugation. HSCs were purified via density gradient centrifugation on 13.14% (w/v) Nycodenz, suspended in Dulbecco's modified Eagle medium containing penicillin/streptomycin and 10% fetal bovine serum/10% horse serum, and plated on uncoated plastic dishes at a density of 1.0 ϫ 10 6 /well (24-well plate).…”

“…21,22 Experiments of the present study demonstrate concentration-dependent stimulation of the synthesis of NO, IL-6, and TNF-␣ by LPS, with significant increases occurring at the LPS concentration of 0.001 g/mL (NO) and 0.01-0.1 g/mL (IL-6 and TNF-␣) (Supplementary Fig. 1; Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/ jpages/0270-9139/suppmat/index.html).…”

“…[14][15][16] Endotoxin, in conjunction with interferon-␥, TNF-␣, and IL-1␤, also stimulates the synthesis of a multifunctional mediator NO in Kupffer cells 17 and hepatocytes. 18 Work from several laboratories, including ours, has demonstrated that endotoxin stimulates synthesis of NO via induction of inducible NO synthase (iNOS), 10,[19][20][21][22] and of TNF-␣ and IL-6 21,22 in both quiescent (normal liver) and activated (fibrotic liver) HSCs. TNF-␣ and IL-6, as well as increased NO (directly or via a potent free radical peroxinitrite), can exert a variety of effects on the hepatic system.…”

Pattern recognition receptors: A contemporary view on liver diseases

“…Stellate cells, the main extracellular matrix-producing cells of the liver, express TLR4, TLR2 and CD14 and respond to TLR2 and 4 ligands by activation of JNK, ERK and NF B and pro-inflammatory cytokines production. [78][79][80][81] The liver also accommodates a variety of immune cells that widely express TLRs. T lymphocytes and NK cells are rich in TLR1,2,4,5 and 9, while B cells express high levels of TLR1,6,7,9 and 10.…”