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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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Ex vivo cytokine responses by blood from LBP−/− mice was  markedly hyporesponsive to exogenously added LPS. Whole blood was  obtained from +/− and −/− mice, pooled, and assessed for LPS responsiveness as described in Materials and Methods. Recombinant LBP was  added to achieve a final concentration of 2 μg/ml. Each data point is expressed as the mean +/− range of two separate experimental wells (each  of which was measured in duplicate). Shown is one of four similar experiments.
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Figure 5: Ex vivo cytokine responses by blood from LBP−/− mice was markedly hyporesponsive to exogenously added LPS. Whole blood was obtained from +/− and −/− mice, pooled, and assessed for LPS responsiveness as described in Materials and Methods. Recombinant LBP was added to achieve a final concentration of 2 μg/ml. Each data point is expressed as the mean +/− range of two separate experimental wells (each of which was measured in duplicate). Shown is one of four similar experiments.

Mentions: We measured the ability of LPS to induce TNF-α secretion in whole blood ex vivo from the LBP−/− animals and their hemizygous littermates. Careful dose response studies of the KO animal revealed that the absence of functional LBP shifted the dose-response relationship of LPS by at least 1,000-fold (Fig. 5). The addition of recombinant LBP to whole blood from the LBP−/− mouse shifted the curve to closely resemble that of the hemizygous mouse (Fig. 5). Thus, blood from mice with the targeted deletion of the LBP gene was markedly hyporesponsive to LPS, presumably as a result of the inability of LPS to rapidly associate with CD14.


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)

Ex vivo cytokine responses by blood from LBP−/− mice was  markedly hyporesponsive to exogenously added LPS. Whole blood was  obtained from +/− and −/− mice, pooled, and assessed for LPS responsiveness as described in Materials and Methods. Recombinant LBP was  added to achieve a final concentration of 2 μg/ml. Each data point is expressed as the mean +/− range of two separate experimental wells (each  of which was measured in duplicate). Shown is one of four similar experiments.
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Related In: Results  -  Collection

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Figure 5: Ex vivo cytokine responses by blood from LBP−/− mice was markedly hyporesponsive to exogenously added LPS. Whole blood was obtained from +/− and −/− mice, pooled, and assessed for LPS responsiveness as described in Materials and Methods. Recombinant LBP was added to achieve a final concentration of 2 μg/ml. Each data point is expressed as the mean +/− range of two separate experimental wells (each of which was measured in duplicate). Shown is one of four similar experiments.
Mentions: We measured the ability of LPS to induce TNF-α secretion in whole blood ex vivo from the LBP−/− animals and their hemizygous littermates. Careful dose response studies of the KO animal revealed that the absence of functional LBP shifted the dose-response relationship of LPS by at least 1,000-fold (Fig. 5). The addition of recombinant LBP to whole blood from the LBP−/− mouse shifted the curve to closely resemble that of the hemizygous mouse (Fig. 5). Thus, blood from mice with the targeted deletion of the LBP gene was markedly hyporesponsive to LPS, presumably as a result of the inability of LPS to rapidly associate with CD14.

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