LPS-binding protein mediates LPS-induced liver injury and mortality in the setting of biliary obstruction

Abstract

It is generally accepted that low levels of lipopolysaccharide (LPS)-binding protein (LBP) augment the cell's response to LPS, whereas high levels of LBP have been shown to inhibit cell responses to LPS. Clinical studies and in vitro work by our group have demonstrated that, in the setting of liver disease, increased or acute-phase levels of LBP may actually potentiate rather than inhibit an overwhelming proinflammatory response. Therefore, in the present studies we sought to determine the role of acute-phase LBP in mediating morbidity and mortality in animals challenged with LPS in the setting of biliary obstruction. Using LBP-deficient mice and LBP blockade in wild-type mice, we demonstrate that high levels of LBP are deleterious in the setting of cholestasis. Following biliary obstruction and intraperitoneal LPS challenge, hepatic injury, hepatic neutrophil infiltration, and mortality were significantly increased in animals with an intact LBP acute-phase response. Kupffer cell responses from these animals demonstrated a significant increase in several inflammatory mediators, and Kupffer cell-associated LBP appears to be responsible for these differences, at least in part. Our results indicate that the role of LBP signaling in inflammatory conditions is complex and heterogeneous, and elevated levels of LBP are not always protective. Increased LBP production in the setting of cholestatic liver disease appears to be deleterious and may represent a potential therapeutic target for preventing overwhelming inflammatory responses to LPS in this setting.

Fig. 1.

Plasma and liver LPS-binding protein (LBP) levels following common bile duct ligation (CBDL). Plasma (A) and liver (B) LBP levels are significantly elevated 4 days following CBDL compared with sham operation, *P < 0.001.

Fig. 2.

Survival following CBDL and LPS challenge. LBP-deficient (LBPKO) mice (n = 21) demonstrate significantly improved survival following delivery of LPS [1 μg/g body weight (gbw)] after CBDL compared with C57BL/6 controls (n = 20), P < 0.005 (A). Similarly, CBDL wild-type mice treated with an inhibitory LBP peptide (n = 10) demonstrate a survival advantage compared with controls treated with saline vehicle alone (n = 10) prior to LPS challenge (1 μg/gbw), P = 0.003 (B).

Fig. 3.

Hepatic transaminase levels following CBDL and LPS challenge. Aspartate aminotransferase (AST; A) and alanine aminotransferase (ALT; B) levels are significantly elevated in C57BL/6 CBDL mice compared with LBPKO CBDL mice 6 h following LPS delivery (1 and 5 μg/gbw), *P < 0.001 and *P < 0.05, respectively.

Fig. 4.

Liver myeloperoxidase (MPO) activity. Myeloperoxidase activity, a measure of neutrophil infiltration, is significantly increased in the livers of C57BL/6 CBDL mice compared with LBPKO CBDL mice following LPS delivery (100 ng/gbw and 1 μg/gbw doses), *P < 0.05.

Fig. 5.

Expression of hepatic TNF-α, IL-6, macrophage inflammatory protein (MIP)-2, and IL-1 receptor antagonist (IL-1ra) mRNA. Hepatic levels of TNF-α (A) mRNA were significantly elevated in the C57BL/6 mice 6 h following the in vivo delivery of 5 μg/gbw LPS, whereas levels of MIP-2 (C) were significantly elevated in the C57BL/6 mice 6 h following delivery of 100 ng/gbw and 1 μg/gbw LPS, *P < 0.05. Hepatic levels of IL-6 (B) and IL-1ra (D) were significantly elevated in the C57BL/6 mice 6 h following the in vivo delivery of 1 and 5 μg/gbw LPS, *P < 0.05.

Fig. 6.

Cell surface expression of LPS receptors. Cell surface expression of the LPS receptors TLR4, CD14, and CD11b/CD18 is equivalent on Kupffer cells isolated from C57BL/6 CBDL (gray trace) and LBPKO CBDL (black trace) mice.

Fig. 7.

Immunofluorescent staining of LBP on C57BL/6 Kupffer cells. Four days following CBDL, Kupffer cells isolated from C57BL/6 mice demonstrate the LBP is present on the cell surface as well within the cell (A), as demonstrated by the confocal microscope images, ×100 magnification. The LBP appears green (stained with Alexa Fluor 488), whereas the nuclei appear blue (stained with DAPI). Kupffer cells isolated from LBPKO CBDL mice (negative control) demonstrate only positive nuclear staining (B).

Fig. 8.

Effect of LBP antibody blockade on Kupffer cell production of TNF-α following LPS stimulation. LBP antibody blockade significantly reduces C57BL/6 CBDL Kupffer cell production of TNF-α in a dose-dependent manner following stimulation of cells with LPS (100 ng/ml), *P < 0.001 compared with 0-μg LBP antibody dose.

Fig. 9.

Effect of LBP antibody blockade on Kupffer cell production of IL-6, MIP-2, and IL-1ra following LPS stimulation. LBP antibody blockade significantly reduces C57BL/6 Kupffer cell production of IL-6 (A), MIP-2 (B), and IL-1ra (C) following stimulation of cells with LPS (100 ng/ml), *P < 0.001 compared with 0-μg LBP antibody dose.