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: 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.
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.
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Mentions: Screening of a murine 129/SVJ genomic library in the P1 cloning vector was performed by Genome Systems, Inc. (St. Louis, MO). A genomic clone was identified using primer pairs to generate PCR products corresponding to portions of the murine LBP 5′ untranslated region (UTR) (5′-CGGGGCCTCTCTTTCCCGC-3′, 5′-CCTGGATGCTCCGTGGGGG-3′) and 3′ UTR (5′-GGGTCTCAGTGGCCACAGC-3′, 5′-CAGGTCTCCCACCCAGTGTTG-3′). Exons 1–3 were sequenced and found to be identical to the murine LBP cDNA sequence (These sequence data are available from EMBL/GenBank/DDBJ under accession number X99347). A 8.4-kb NheI–NdeI subclone was used to construct the targeting vector. A 1.4-kb SpeI fragment was replaced with the neomycin phosphotransferase gene (neo) in the opposite transcriptional orientation from the LBP gene. Replacement of LBP sequences with the neo cassette deleted a portion of the 5′ UTR, the start codon and subsequent 40 amino acids, and introduced a new EcoRV restriction site. A map of the 5′ portion of the LBP gene and the targeting vector is shown in Fig. 1.