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Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responses ex vivo, whereas in vivo responses remain intact.

Wurfel MM, Monks BG, Ingalls RR, Dedrick RL, Delude R, Zhou D, Lamping N, Schumann RR, Thieringer R, Fenton MJ, Wright SD, Golenbock D - J. Exp. Med. (1997)

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.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York 10021, USA.

ABSTRACT
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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Southern blotting analysis of DNA from mutant LBP−/−,  hemizygous LBP+/−, and homozygous LBP+/+ F1 littermates. Southern  blots of tail-tip DNA of mice from the first heterozygote/heterozygote  crossing are shown. The mutant allele is ∼6 kb and the wild-type allele is  11 kb.
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Figure 2: Southern blotting analysis of DNA from mutant LBP−/−, hemizygous LBP+/−, and homozygous LBP+/+ F1 littermates. Southern blots of tail-tip DNA of mice from the first heterozygote/heterozygote crossing are shown. The mutant allele is ∼6 kb and the wild-type allele is 11 kb.

Mentions: An ES cell clone that underwent homologous recombination was expanded and injected into C57BL/6J blastocysts. The resulting chimeric animals were backcrossed to C57BL/6J (Jackson Laboratories, Bar Harbor, ME) mice. Germline transmission was identified by Southern blot analysis of tail DNA. Hemizygous animals were crossed and homozygous offspring were identified by Southern blot as shown in Fig. 2.


Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responses ex vivo, whereas in vivo responses remain intact.

Wurfel MM, Monks BG, Ingalls RR, Dedrick RL, Delude R, Zhou D, Lamping N, Schumann RR, Thieringer R, Fenton MJ, Wright SD, Golenbock D - J. Exp. Med. (1997)

Southern blotting analysis of DNA from mutant LBP−/−,  hemizygous LBP+/−, and homozygous LBP+/+ F1 littermates. Southern  blots of tail-tip DNA of mice from the first heterozygote/heterozygote  crossing are shown. The mutant allele is ∼6 kb and the wild-type allele is  11 kb.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199164&req=5

Figure 2: Southern blotting analysis of DNA from mutant LBP−/−, hemizygous LBP+/−, and homozygous LBP+/+ F1 littermates. Southern blots of tail-tip DNA of mice from the first heterozygote/heterozygote crossing are shown. The mutant allele is ∼6 kb and the wild-type allele is 11 kb.
Mentions: An ES cell clone that underwent homologous recombination was expanded and injected into C57BL/6J blastocysts. The resulting chimeric animals were backcrossed to C57BL/6J (Jackson Laboratories, Bar Harbor, ME) mice. Germline transmission was identified by Southern blot analysis of tail DNA. Hemizygous animals were crossed and homozygous offspring were identified by Southern blot as shown in Fig. 2.

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.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York 10021, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus