Kupffer cell activation by lipopolysaccharide in rats: Role for lipopolysaccharide binding protein and toll-like receptor 4

Abstract

Lipopolysaccharide (LPS) binding protein (LBP) is a keyEndogenous lipopolysaccharides (LPS) have been implicated as a cofactor in promoting liver injury in many models of liver injury, including alcoholic hepatitis. 1,2 In the Tsukomoto and French model of rat alcoholic hepatitis, 3 the degree of liver injury is diminished by treatment with either antibiotics, lactobacillus, or polymixin, all of which decrease endogenous LPS. 4,5 In this model, Kupffer cells, when activated by LPS, play a prominent role in promoting liver injury. 6 Despite its potentially critical importance, the molecular mechanism by which Kupffer cells are activated by LPS remains largely unknown.In peripheral blood monocytes, the pathway of LPS activation has been recently delineated. In serum, LPS binds to LPS-binding protein (LBP), which is a 60-kd acute-phase protein produced by hepatocytes. 7,8 This LPS-LBP complex then binds to membrane CD14 resulting in cell activation, nuclear translocation of NF-␤, and production of cytokines such as TNF-␣. 9,10 The critical importance of LBP during in vivo responses to LPS and gram-negative bacteria is clearly shown by the inability of LBP knock-out mice to fight intraperitoneal infections 11 as well as the ability of anti-LBP monoclonal antibodies to prevent lethality in the LPS/ galactosamine model of endotoxemia. 12 Multiple lines of evidence suggest that the mechanisms by which LPS activates Kupffer cells may differ from those found in blood monocytes. Some authors have suggested that LPS activation in Kupffer cells is not mediated via the LBP/CD14 pathway hypothesized for blood monocytes. 13,14 Support for this idea stems from reports showing relatively low levels of CD14 expression in resting Kupffer cells compared with RAW 264.7 cells (murine macrophage cell line) and peritoneal macrophages. 15 Furthermore, in some studies, LPS activation of Kupffer cells, unlike that in peripheral blood monocytes, is not augmented by the addition of serum, suggesting that the LBP found in serum may act differently in Kupffer cells. 13,14 Because serum contains multiple factors that may alter LPS activation, we sought to focus on LBP' s role in Kupffer cell activation by performing experiments using recombinant rat LBP. In addition, because Kupffer cells express relatively low levels of CD14 in the resting state, we sought to examine the role of another candidate LPS receptor, the Toll-like receptor 4 (Tlr 4), in mediating the effects of LBP on LPS activation of Kupffer cells. MATERIALS AND METHODSReagents. LPS from Escherichia coli (055:B5) was purchased from Sigma (St Louis, MO), and pronase was obtained from Boehringer Mannheim (Indianapolis, IN).Recombinant Rat LBP. Recombinant rat LBP was produced using the baculovirus expression system. Briefly, the full-length rat LBP complementary DNA (cDNA) 16 was cloned in frame into pBluebacHis2c (Invitrogen, Carlsbad, CA) and used in conjunction with the linearized defective baculovirus DNA, Bac-N-Blue (Invitrogen, Carlsbad, CA) to cotransfect Sf9 insect cells. Re...

Type I, but not type II, interferon is critical in liver injury induced after ischemia and reperfusion

“…29 Although our inability to extend the conclusion on LPS response from bone marrow macrophages directly to liver KC represents a limitation, it has recently been shown that KC do express functional TLR4 (including CD14 and MD2) and respond to LPS in a fashion similar to other macrophage populations. 30,31 Conversely, the use of in vitro macrophage/hepatoma cell culture systems broaden the significance of our findings and allowed study of TLR4-dependent events in well-defined liver cell substitutes rather than in native liver cells that have been exposed to prolonged and often harmful ex vivo separation procedures. Although macrophage-hepatoma cell culture experiments support our hypothesis, KC-hepatocyte interactions in vivo still need to be evaluated in future studies.…”

Cutting Edge: TLR4 Activation Mediates Liver Ischemia/Reperfusion Inflammatory Response via IFN Regulatory Factor 3-Dependent MyD88-Independent Pathway

“…IL-1, IL-6, and TNF-␣ enhance liver TLR2, but not TLR4 expression (10). Functionally, Kupffer cells are capable of responding to LPS stimulation (12). However, whether TLR4 expression in hepatocytes is sufficient to respond to TLR ligands remains uncertain.…”

Aggravation of Galactosamine Hepatotoxicity by Albumin in Rats

“…LPS has been reported to increase the plasma LBP level by promoting its synthesis in the liver, intestine, and lung 30 . LBP transfers LPS as LPS/LBP complexes 31 to cell surface CD14 presented on the cells of myeloid lineage 32 , and thus LBP activates monocytes and tissue macrophages 31,33 to produce TNF-α 34 . However, LBP has been reported to induce different responses depending on its concentration in vitro.…”