Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responses ex vivo, whereas in vivo responses remain intact.
Bottom Line: Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS.Despite these striking in vitro findings, no significant differences in TNF-alpha levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS.These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.
Affiliation: The Rockefeller University, New York 10021, USA.
Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-alpha. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-alpha levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.
Related in: MedlinePlus
Mentions: Initial restriction mapping of the murine LBP gene demonstrated that it is at least 14 kb in length. Since the gene was too large to delete in its entirety, we constructed a vector to delete the first exon, including the translational start site, and part of the first intron. PCR primers derived from the deleted region of the 5′ UTR to the 3′ UTR amplified the predicted products from RNA derived from wild-type mice but not KO mice, thus confirming the deletion (data not shown). To determine if the remaining portion of the gene was transcribed in KO animals, RNA was prepared and subjected to reverse transcriptase PCR analysis using primers that were not in the region of the gene deletion. Liver mRNA from LBP−/− mice expressed only a small fraction of LBP transcript in comparison to wild-type mice and no LBP mRNA was detected in kidneys from −/− mice (Fig. 3). These data confirmed that the KO mouse expresses very low levels of mutant LBP transcript. Even if the mutant mRNA were translated successfully, the resultant protein would be truncated and probably expressed at very low levels.