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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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Plasma from the knock-out mice does not contain functional LBP. (a) LPS-coated plastic was incubated with plasma from wild-type LBP+/+, hemizygous LBP+/−, and homozygous LBP−/− mice. LPS-bound LBP was detected by incubating washed wells with polyclonal anti-LBP serum as described  in Materials and Methods. No detectable LBP remained bound to the plastic in plasma from the KO animals. (b) Plasma from LBP+/− animals was compared to plasma from homozygous LBP−/− animals for its ability to mediate transfer of LPS to sCD14. B-LPS plus LBP+/− plasma plus sCD14 (•), B-LPS  plus LBP−/− plasma plus sCD14 (▴), B-LPS plus LBP+/− plasma (○), B-LPS plus LBP−/− plasma (▵), B-LPS alone (□). The KO plasma had virtually  no ability to transfer LPS to sCD14. Addition of recombinant LBP to LBP−/− plasma restored activity (not shown). Results are representative of four separate experiments.
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Figure 4: Plasma from the knock-out mice does not contain functional LBP. (a) LPS-coated plastic was incubated with plasma from wild-type LBP+/+, hemizygous LBP+/−, and homozygous LBP−/− mice. LPS-bound LBP was detected by incubating washed wells with polyclonal anti-LBP serum as described in Materials and Methods. No detectable LBP remained bound to the plastic in plasma from the KO animals. (b) Plasma from LBP+/− animals was compared to plasma from homozygous LBP−/− animals for its ability to mediate transfer of LPS to sCD14. B-LPS plus LBP+/− plasma plus sCD14 (•), B-LPS plus LBP−/− plasma plus sCD14 (▴), B-LPS plus LBP+/− plasma (○), B-LPS plus LBP−/− plasma (▵), B-LPS alone (□). The KO plasma had virtually no ability to transfer LPS to sCD14. Addition of recombinant LBP to LBP−/− plasma restored activity (not shown). Results are representative of four separate experiments.

Mentions: To determine if blood contained a functional mutant LBP molecule, we analyzed plasma from KO mice for LBP-like activity. The ability of LBP to bind LPS and to form a stable complex has been well described (9, 16). We incubated plasma from the KO mouse with LPS-coated plastic, washed the wells with buffer, and developed the plate with a polyclonal antiserum against murine LBP. Although hemizygous mice expressed wild-type levels of LBP, their homozygous gene-targeted littermates had no detectable LBP (Fig. 4 a).


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)

Plasma from the knock-out mice does not contain functional LBP. (a) LPS-coated plastic was incubated with plasma from wild-type LBP+/+, hemizygous LBP+/−, and homozygous LBP−/− mice. LPS-bound LBP was detected by incubating washed wells with polyclonal anti-LBP serum as described  in Materials and Methods. No detectable LBP remained bound to the plastic in plasma from the KO animals. (b) Plasma from LBP+/− animals was compared to plasma from homozygous LBP−/− animals for its ability to mediate transfer of LPS to sCD14. B-LPS plus LBP+/− plasma plus sCD14 (•), B-LPS  plus LBP−/− plasma plus sCD14 (▴), B-LPS plus LBP+/− plasma (○), B-LPS plus LBP−/− plasma (▵), B-LPS alone (□). The KO plasma had virtually  no ability to transfer LPS to sCD14. Addition of recombinant LBP to LBP−/− plasma restored activity (not shown). Results are representative of four separate experiments.
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Related In: Results  -  Collection

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Figure 4: Plasma from the knock-out mice does not contain functional LBP. (a) LPS-coated plastic was incubated with plasma from wild-type LBP+/+, hemizygous LBP+/−, and homozygous LBP−/− mice. LPS-bound LBP was detected by incubating washed wells with polyclonal anti-LBP serum as described in Materials and Methods. No detectable LBP remained bound to the plastic in plasma from the KO animals. (b) Plasma from LBP+/− animals was compared to plasma from homozygous LBP−/− animals for its ability to mediate transfer of LPS to sCD14. B-LPS plus LBP+/− plasma plus sCD14 (•), B-LPS plus LBP−/− plasma plus sCD14 (▴), B-LPS plus LBP+/− plasma (○), B-LPS plus LBP−/− plasma (▵), B-LPS alone (□). The KO plasma had virtually no ability to transfer LPS to sCD14. Addition of recombinant LBP to LBP−/− plasma restored activity (not shown). Results are representative of four separate experiments.
Mentions: To determine if blood contained a functional mutant LBP molecule, we analyzed plasma from KO mice for LBP-like activity. The ability of LBP to bind LPS and to form a stable complex has been well described (9, 16). We incubated plasma from the KO mouse with LPS-coated plastic, washed the wells with buffer, and developed the plate with a polyclonal antiserum against murine LBP. Although hemizygous mice expressed wild-type levels of LBP, their homozygous gene-targeted littermates had no detectable LBP (Fig. 4 a).

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