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...
Upregulation of CD14 in Kupffer cells has been implicated in the pathogenesis of several forms of liver injury, including alcoholic liver disease. However, it remains unclear whether CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using several complementary approaches. First, we isolated Kupffer cells from human livers and studied the effects of anti-CD14 antibodies on LPS activation of these cells. Kupffer cells were incubated with increasing concentrations of LPS in the presence and absence of recombinant human LPS binding protein (LBP). With increasing concentrations of LPS, human Kupffer cell tumor necrosis factor-alpha (TNF-alpha) production (a marker for Kupffer cell activation) increased in a dose-dependent manner in the presence and absence of LBP. In the presence of anti-human CD14 antibodies, the production of TNF-alpha was significantly diminished. Second, we compared LPS activation of Kupffer cells isolated from wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice produced significantly less TNF-alpha in response to the same amount of LPS. Together, these data strongly support a critical role for CD14 in Kupffer cell responses to LPS.
This study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.
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