Lipopolysaccharide (LPS)-binding Protein Inhibits Responses to Cell-bound LPS*
Patricia A. Thompson
,
Peter S. Tobias
,
Suganya Viriyakosol ¶,
Theo N. Kirkland ¶ and
Richard L. Kitchens
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From the
Department of Internal Medicine, Division
of Infectious Diseases, University of Texas Southwestern Medical Center,
Dallas, Texas 75390-9113, the
Department of
Immunology, Scripps Research Institute, LaJolla, California 92037, and the
¶Department of Pathology, University of
California San Diego School of Medicine, the Veterans Affairs San Diego
Healthcare System, San Diego, California 92161
Received for publication, March 21, 2003
, and in revised form, May 9, 2003.
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ABSTRACT
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Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant
that may play a dual role in vivo, both potentiating and decreasing
cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate
cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit
cell responses to LPS. One inhibitory mechanism involves the ability of LBP to
neutralize LPS by transferring it to plasma lipoproteins, whereas other
inhibitory mechanisms, such as the one described here, do not require
exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses
to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell
surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the
ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable
LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS
in 10 min, this was not accompanied by extensive release of the LPS from the
cells. The cross-linking data suggest that much of the LPS that remained bound
to the cells was associated with LBP. The ability of LBP to inhibit cell
responses could not be explained by its effect on LPS internalization, because
LBP did not significantly increase the internalization of the cell-bound LPS.
In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS
from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP
can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the
Toll-like receptor 4-MD-2 signaling receptor.
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INTRODUCTION
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Lipopolysaccharide
(LPS1; endotoxin), an
abundant component of the outer membrane of Gram-negative bacteria, is one of
the most potent of the known bacterial agonists in animal host cells. Whereas
LPS recognition benefits the host by sensing the presence of bacteria and
mobilizing defense mechanisms, an exaggerated response to LPS may contribute
to the harmful sequelae of severe sepsis, which include coagulation disorders,
organ failure, shock, and death. The host, therefore, has numerous mechanisms
that down-regulate responses to LPS and remove it from the circulation and
tissues
(17).
The potency of LPS is due to the sensitivity of the host recognition
system, which requires the Toll-like receptor 4 (Tlr4)
(8) signaling receptor. At
least three LPS-binding proteins (LBP
(9), CD14
(10), and MD-2
(11)) are required for
sensitive Tlr4-mediated LPS recognition. Plasma LBP potentiates LPS
recognition by transferring it from bacterial membranes
(6) to CD14
(12,
13), which is expressed as a
glycosylphosphatidylinositol-anchored protein on the surfaces of myeloid
lineage cells (mCD14) and as an anchorless plasma protein, soluble CD14
(sCD14) (14,
15). CD14 presents LPS to
Tlr4, presumably by transferring the LPS to MD-2, an accessory protein that is
constitutively associated with the extracellular domain of Tlr4
(16). MD-2 binds LPS with high
affinity (17), and it is
required for Tlr4 signaling
(11). Although normal
expression of the Tlr4 and MD-2 are too low for direct measurements of LPS
binding to the receptor complex, indirect evidence of ligand-receptor binding
has been provided by the ability of structurally different forms of Tlr4
(18,
19) or MD-2
(20,
21) to discriminate between
differences in lipid A structure. Direct evidence of LPS binding to Tlr4/MD-2
was provided by LPS cross-linking experiments in cells that overexpressed
these proteins (22).
LBP and sCD14 can both potentiate and down-regulate responses to LPS. sCD14
was originally described as an LPS inhibitor
(23), and other studies have
described inhibitory effects of high concentrations of sCD14 under various
conditions (24,
25). Recent work from our
laboratory has shown that in plasma, sCD14 decreases monocyte responses to LPS
by transferring cell-bound LPS to plasma lipoproteins
(26).
LBP can prevent lethal shock caused by LPS or Gram-negative bacteria when
administered intraperitoneally in mice
(27). High acute phase
concentrations of LBP in the plasma of septic humans decrease monocyte
responses to LPS (28). The
inhibitory mechanism of LBP can be explained in part by its ability to
neutralize the bioactivity of LPS by transferring it to plasma lipoproteins
(6,
29). Other inhibitory
mechanisms are suggested by the ability of LBP to inhibit cell responses to
LPS in serum-free medium (27,
28). Possible mechanisms might
include the formation of large LPS·LBP complexes that are internalized
by mCD14 but that contribute little to LPS signaling
(30). Here we report that LBP
can inhibit LPS signaling even after LPS monomers have bound to mCD14.
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EXPERIMENTAL PROCEDURES
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Reagents and CellsRecombinant human LBP (C-terminal His
tag) was expressed in a baculovirus system in SF-9 cells in SF-900 II SFM
(Invitrogen). The protein was purified to homogeneity on a His-TrapTM
nickel column (Amersham Biosciences). Recombinant human sCD14 and soluble MD-2
(sMD-2) were produced by baculovirus expression as previously described
(17,
26). The recombinant proteins
did not stimulate cells when added alone at the indicated concentrations.
THP-1 cells (provided by LiWu Li, Wake Forest University, Winston-Salem, NC)
were cultured in 0.05 µM 1,25-dihydroxyvitamin D3
(VD3) for 34 days to induce mCD14 expression (THP-1
(VD3)). In experiments in which IL-1
was used as an agonist,
the cells were cultured in 106 M
prostaglandin E2 (PGE2) during the last 24 h of culture
in VD3 (THP-1 (VD3-PGE2)). PGE2
and VD3 induced responsiveness of the cells to IL-1
without
inhibiting responsiveness to LPS. In some experiments (Figs.
3 and
4B), mCD14 expression
was enhanced by stable transfection of THP-1 cells with human CD14 cDNA as
previously described (31), and
the cells were also differentiated by culturing them in VD3 as
described above. Peripheral blood mononuclear cells were isolated from normal
human blood as previously described
(26) with the approval of the
Institutional Review Board of UT Southwestern Medical Center. HEK 293 cells
(ATCC) were transiently transfected with human CD14 cDNA (pRcRSV-CD14)
(31) using Fugene 6 (Roche
Applied Science). Anti-human CD14 monoclonal antibodies, 60bca and 63D3, were
purified from the culture supernatants of hybridomas obtained from the ATCC.
60bca was covalently bound to agarose beads using CarbolinkTM from
Pierce. Affinity-purified rabbit polyclonal anti-human LBP antibody, K1970602,
was a gift of XOMA Corp. (Berkeley, CA). IgG fractions of goat polyclonal
antibodies to recombinant human soluble CD14 (number 4-2051-5) and recombinant
human LBP (number 4-1855-9) were produced in the Tobias laboratory.
Peroxidase-conjugated donkey anti-goat IgG was obtained from Jackson
Immunoresearch (West Grove, PA). All other reagents were from Sigma unless
otherwise stated.

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FIG. 3. LBP does not strongly influence the release or internalization of
cell-bound LPS. CD14-transfected THP-1 (VD3) cells were
preincubated with [3H]LPS-sCD14 complexes (100 ng of LPS/ml)
(A) or Alexa-LPS·sCD14 complexes (200 ng of LPS/ml)
(B) in the absence of LBP for 5 min to allow maximal binding of
monomeric LPS to mCD14. The cells were then washed to remove unbound LPS and
were incubated for the indicated times in SFM or in SFM containing 3 µg/ml
rLBP (SFM + LBP). Total cell-associated [3H]LPS and cell-associated
[3H]LPS that was not released by treatment of the cells with
proteinase K (internalized LPS) are shown in A. B, total
cell-associated Alexa-LPS and cell-associated Alexa-LPS that was not quenched
by trypan blue (internalized LPS). The [3H]LPS (A) and the
mean fluorescence intensities of Alexa-LPS, measured by flow cytometry
(B) were expressed as the percentage of the total LPS that initially
bound to the cells. Each data point represents the mean ± range of two
experiments that were performed in duplicate.
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FIG. 4. LBP can remove cell-bound LPS from mCD14. 125I-ASD-LPS
was bound to CD14-transfected HEK 293 cells (A) or CD14-transfected
THP-1 (VD3) cells (B) by incubating the cells with
preformed 125I-ASD·LPS·sCD14 complexes (120200
ng of LPS/ml) for 3 min at 37 °C. The cells were washed and incubated for
10 min in SFM or in SFM containing 3 µg/ml rLBP (SFM + LBP) and were then
exposed to UV light to induce cross-linking of the
125I-ASD·LPS to protein. The cells were then washed, lysates
were prepared, and mCD14 (CD14 i.p.) and LBP (LBP i.p.) were
sequentially immunoprecipitated from the lysates. The immunoprecipitated
125I-proteins were separated by SDS-PAGE, and the radioactivity in
the CD14 and LBP bands was quantitated by phosphor imaging. To measure the
inhibitory effect of LBP, the average CD14 radioactivity in lanes 3
and 4 is expressed as the percentage of the average CD14
radioactivity in lanes 1 and 2. The experiments were
repeated twice with similar results. Similar results were obtained when mCD14
was immunoprecipitated with an antibody (63D3) that binds to a different CD14
epitope (data not shown).
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LPS[3H]LPS was biosynthetically labeled in the
fatty acyl chains in Escherichia coli LCD25 (Ra chemotype)
(32). 125I-ASD-LPS
was made by derivatization of RcLPS (Salmonella minnesota R5) with
sulfosuccinimidyl-2-[p-azidosalicylamido]ethyl-13'-dithiopropionate
(SASD) (Pierce), a cleavable, photoactivatable cross-linker, as previously
described (33). The
stimulatory activity of the 125I-ASD-LPS was tested in THP-1
(VD3) cells and found to be similar to that of the underivatized
RcLPS. Each LPS preparation stimulated cytokine production at threshold
concentrations of 0.030.1 ng of LPS/ml in the presence of LBP (0.1
µg/ml) or as preformed LPS-sCD14 complexes (data not shown). Fluorescent
LPS (Alexa Fluor488TM-LPS derived from E. coli 055:B5 LPS) was
obtained from Molecular Probes, Inc. (Eugene, OR) and is referred to here as
Alexa-LPS. Protein contamination was not found in any of the LPS preparations
on silver-stained SDS-PAGE gels.
LPS-Cell Binding and StimulationLPS-cell binding was
performed in the absence of LBP using preformed LPS·sCD14 complexes
(34). For cell stimulation
assays, the cells (7 x 105 cells/0.1 ml) were incubated with
LPS-sCD14 at 1 ng of LPS/ml for 2 min at 37 °C. The cells were then washed
in cold RPMI 1640 medium to remove unbound LPS and were incubated in
serum-free medium (SFM) (RPMI 1640, 20 mM HEPES, pH 7.4, 0.1 mg/ml
bovine serum albumin) containing the indicated proteins for 2 h at 37 °C
with brief mixing at 2-min intervals. Cytokines were measured in culture
supernatants by enzyme-linked immunosorbent assay (BD Pharmingen, San Diego,
CA). The cell-bound LPS stimulated the production of
44 ng of IL-8/ml,
5.5 ng of tumor necrosis factor-
/ml, and 1.7 ng of IL-1
/ml during
2-h incubations, whereas unstimulated cells produced less than 2% of those
cytokine levels.
The release of cell-bound [3H]LPS was measured by incubating the
cells with 100 ng/ml [3H]LPS in LPS·sCD14 complexes for
25 min at 37 °C, washing with cold SFM to remove unbound LPS, and
incubating the cells in SFM at 37 °C for the indicated times. Released and
cell-associated [3H]LPS were measured as previously described
(35). LPS internalization was
measured by protease protection of [3H]LPS and by surface quenching
of fluorescent LPS with trypan blue as previously described
(31,
34). Briefly, cell
surface-bound [3H]LPS was removed by incubating the cells in 0.02%
proteinase K for 30 min at 4 °C, or the fluorescence of cell surface-bound
Alexa-LPS was quenched by suspending the cells in trypan blue and measuring
the mean fluorescence intensity by flow cytometry using a FACScanTM (BD
Biosciences).
In LPS cross-linking experiments, 125I-ASD-LPS was prebound to
rsCD14, the mixture was then incubated in SFM with the indicated soluble
proteins or with cells at 37 °C, and the mixtures were then exposed to UV
light to covalently cross-link the 125I moiety to the associated
proteins. After cross-linking the LPS to cellular proteins, the cells (
4
x 106 cells/0.5 ml) were washed and lysed in 50 µl of
lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2
mM EDTA, 1% Triton X-100, 20 mM octyl glucoside, 5
mM iodoacetamide, 1 mM benzamidine, 0.25 mM
phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, and 1
µg/ml each of pepstatin A, leupeptin, and aprotinin) for 30 min at 4 °C
and centrifuged to remove insoluble material. CD14 and LBP were
immunoprecipitated sequentially by incubating the lysate supernatants with 12
µl of 60bca-agarose beads for 2 h at 4 °C with brief agitation (3 s) at
2-min intervals in an Eppendorf ThermomixerTM (Brinkmann Instruments).
After removing the beads, LBP was immunoprecipitated from the lysates by
incubating them with 2 µg of rabbit anti-human LBP for 1 h followed by 12
µl of protein A-Sepharose for 1 h as described above. The beads were then
washed and eluted in SDS sample buffer as previously described
(33). After reductive cleavage
of LPS from the ASD group with dithiothreitol, the 125I-proteins
were separated on 11% SDS-PAGE gels. 125I radioactivity was
quantitated by exposing the dried gel to a phosphor screen and measuring the
radioactivity in a Cyclone PhosphorImager System (Packard Instrument Co.).
Background activity, defined by equally sized control areas of the gel, was
subtracted using the software provided by the manufacturer. In experiments in
which LPS was cross-linked to soluble proteins, autoradiography was performed
by exposing the gels to x-ray film.
To test the specificity of the immunoprecipitations, CD14 and LBP were
immunoprecipitated as described above using specific antibodies or control
antibodies (an irrelevant isotype-matched (IgG1) mAb bound to beads or
nonimmune rabbit IgG and protein A-Sepharose, respectively). After separating
the proteins by SDS-PAGE, they were transferred to Immobilon-P membranes
(Millipore Corp., Bedford, MA) according to the manufacturer's instructions.
The membranes were blocked with 1% fish gelatin in PBS plus 0.05% Tween 20,
and the proteins were detected with goat anti-human CD14 (7 µg/ml) or goat
anti-human LBP (3 µg/ml) followed by peroxidase-conjugated donkey anti-goat
IgG. The blots were developed with ECL reagents (Amersham Biosciences) and
exposed to x-ray film. The CD14 and LBP bands were found at their expected
molecular sizes (
55 and 60 kDa, respectively), whereas no bands were
detected in control antibody immunoprecipitates (data not shown).
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RESULTS
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To determine whether LBP can inhibit cell responses after LPS-mCD14 binding
has occurred, we incubated LPS-sCD14 complexes with mCD14-expressing THP-1
cells for 2 min at 37 °C. We previously found that nearly maximal binding
of LPS monomers to mCD14 occurs under these incubation conditions
(34). After washing to remove
unbound LPS, we found that incubating the cells with LBP inhibited cytokine
production. As shown in Fig. 1,
50% inhibition of cytokine production occurred at LBP concentrations of
12 µg/ml. In experiments not shown, LBP also inhibited responses to
cell-bound LPS in normal human monocytes in peripheral blood mononuclear cell
mixtures with similar potency. Because the LPS receptor (Tlr4) and the IL-1
receptor use many of the same intracellular signaling proteins to induce the
expression of inflammatory cytokine genes, we stimulated the cells with
IL-1
to test whether the inhibitory effect of LBP is LPS-specific. As
shown in Table I, the cells
produced cytokines in response to both LPS and IL-1
, and LBP inhibited
responses only to LPS.

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FIG. 1. LBP inhibits responses to cell-bound LPS. LPS monomers were prebound
to THP-1 (VD3) cells by incubating the cells with LPS-rsCD14
complexes (1 ng LPS/ml) in SFM for 2 min at 37 °C. The cells were washed
and incubated for 2 h in SFM with or without the indicated concentrations of
rLBP. Cytokines were measured in the culture supernatants by enzyme-linked
immunosorbent assay. The means ± range for two experiments are shown as
percentages of control (no LBP).
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TABLE I LBP does not inhibit cell responses to IL-1
THP-1 (VD3-PGE2) cells were preincubated with LPS as
described in the legend to Fig.
1 or with SFM. The cells were washed and incubated for 2 h in SFM
or in SFM containing rIL-1 (2 ng/ml) in the presence or absence of 3
µg/ml rLBP. The cytokine content of the culture supernatant is expressed as
ng/ml and as a percentage of control (% of Ctrl) production obtained in the
absence of LBP. The data are shown as mean ± range of two experiments
that were each performed in duplicate. Cytokines produced by unstimulated
cells were less than 1% of the indicated control levels.
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As shown in Fig. 2, LBP had
its strongest inhibitory effect when added immediately after LPS-cell binding,
and delaying the addition of LBP caused a progressive loss of its ability to
inhibit the responses. Under the conditions of our 2-h incubation, LBP
measurably inhibited IL-8 and tumor necrosis factor-
production when
its addition was delayed for up to 30 min, and it inhibited IL-1
production when added up to 1 h after LPS-cell binding had occurred.

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FIG. 2. Effect of the delayed addition of LBP on responses to cell-bound
LPS. LPS was prebound to THP-1 (VD3) cells as described in the
legend to Fig. 1. The cells
were washed and incubated in SFM to which rLBP was added either immediately
(time 0) or at the indicated times after the incubation had begun. The total
incubation time measured from time 0 in all samples was 120 min. Cytokines
were measured in the culture supernatants by enzyme-linked immunosorbent
assay. The means ± S.D. for three experiments are shown as percentages
of control.
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We next asked whether LBP could enhance the release or internalization of
cell-bound LPS. LPS binding and internalization were measured by two
independent methods (31,
34). In the first
(Fig. 3A), cell
surface-bound [3H]LPS was measured by the ability of proteinase K
to release the [3H]LPS from the cells. Protection of
cell-associated [3H]LPS from proteolytic release may either reflect
its internalization or its transfer to a protease-resistant site on the cell
surface. Therefore, we use a second method in which the binding and
internalization of a fluorescent derivative of LPS were measured by flow
cytometry; trypan blue was used to quench the fluorescence of the
surface-exposed LPS (Fig.
3B). The results showed that whereas LBP slightly
increased the release of LPS from the cells, most of the LPS remained
associated with the cells after they were exposed to 3 µg/ml of LBP, an LBP
concentration that produced a 6075% inhibition of the cell response
(Fig. 1 and
Table I). Whereas the
LBP-induced reduction of cell-associated Alexa-LPS
(Fig. 3B) was greater
than that of [3H]LPS (Fig.
3A), it is unclear whether more Alexa-LPS was actually
released from the cells or whether the binding of LBP to the cell-associated
Alexa-LPS caused a slight reduction in its fluorescence intensity. In any
case, LBP did not significantly increase the rate of release of
cell-associated Alexa-LPS except at the earliest time point. Also shown in
Fig. 3, LBP did not
significantly increase the internalization of cell-bound LPS.
To determine whether the cell-associated LPS remained bound to mCD14 after
the cells were incubated with LBP, we derivatized LPS with a radioiodinatable,
photoactivatable cross-linker (SASD) and measured the effect of LBP on the
ability of cell-bound 125I-ASD-LPS to cross-link to mCD14. The
results shown in Fig. 4 suggest
that LBP dramatically increased the dissociation of cell-bound
125I-ASD-LPS from mCD14 in both CD14-transfected HEK 293 cells and
THP-1 cells. The ability of LBP to remove LPS from mCD14 occurred rapidly; 60%
inhibition of cross-linking occurred after the cells were incubated with LBP
for 5 min (data not shown), and 6475% inhibition occurred after 10 min
(Fig. 4). Immunoprecipitation
of LBP from lysates of the LBP-treated cells revealed that a significant
amount of cell-bound LPS was associated with LBP
(Fig. 4). No labeled protein
band was immunoprecipitated from cells that were not incubated with LBP (data
not shown). The specificities of the immunoprecipitations were demonstrated by
Western blotting (see "Experimental Procedures"). In other
experiments not shown, we confirmed that the 125I-labeled CD14
immunoprecipitates were not derived from sCD14 that may have bound to the
cells. When we cross-linked 125I-ASD-LPS to sCD14 and ran this
product on the gel beside the 125I-labeled immunoprecipitate from
the cell lysate, the 125I-sCD14 was smaller (
46 kDa) than the
125I-mCD14 band (
55 kDa), as expected.
We could not measure LPS binding to MD-2 and Tlr4, which are present in
very low abundance on the cell surface. We therefore used soluble recombinant
proteins in a cell-free assay to measure the impact of LBP on the transfer of
125I-ASD-LPS from CD14 to MD-2. As shown in
Fig. 5, sCD14 (lane 1)
transferred LPS to sMD-2 (lane 2). LBP inhibited LPS transfer from
sCD14 to sMD-2 (lanes 3 and 4) in keeping with the previous
finding that LBP can inhibit the binding of free LPS to sMD-2
(17). When LBP was added after
LPS had already been transferred to sMD-2 (lanes 5 and 6),
LBP removed a significant amount of the LPS from sMD-2 (lanes 7 and
8). Taken together, our data support the hypothesis that LBP may
inhibit the transfer of LPS from mCD14 to the Tlr4-MD-2 signaling
receptor.

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FIG. 5. LBP inhibits LPS transfer from sCD14 to sMD-2.
125I-ASD-LPS was preincubated with a 1.5-fold molar excess of sCD14
to produce LPS-sCD14 complexes. The complexes (4.2 pmol of LPS and 6.2 pmol of
sCD14 in 25 µl) were then incubated for 10 min in SFM containing the
indicated proteins (shown as pmol/25 µl). In lanes 58, the
LPS·sCD14 complexes were preincubated with sMD-2 for 10 min followed by
LBP for an additional 10 min. The 125I-ASD·LPS was then
covalently cross-linked to the binding proteins by exposing the samples to UV
light. The 125I-proteins were then separated on SDS-PAGE after
reductive cleavage of the LPS, and the autoradiograph was produced by exposing
the dried gel to film. The experiment was repeated with similar results.
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DISCUSSION
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Our data reveal a new mechanism by which LBP can inhibit cell responses to
LPS. LBP rapidly removed up to 75% of cell-bound LPS from mCD14
(Fig. 4) and a significant
amount of LPS from MD-2 (Fig.
5), suggesting that LBP attenuates signal responses by interfering
with LPS interactions with the extracellular domains of mCD14 and the
Tlr4·MD-2 receptor complex. Whereas our data show that LBP promoted
some release of the cell-associated LPS into the medium
(Fig. 3), it seems unlikely
that this low percentage of LPS release could account for the strong
inhibitory effect. The ability of LBP to rapidly and almost completely remove
cell-bound LPS from mCD14 provides a more likely explanation for the impact of
LBP on cellular responses. Our data also suggest that a significant amount of
the cell-bound LPS forms a complex with LBP that remains associated with the
cells. It is unclear if the LPS·LBP complexes remain bound to mCD14 or
if they move to another membrane location. If the complexes remain associated
with mCD14, their inability to stimulate the cells is consistent with the
previous finding that most ternary LPS·LBP·mCD14 complexes do
not trigger signal responses and are eventually internalized
(30). If the LPS-LBP complexes
become bound to another membrane structure, they may not be able to induce
signaling, because LPS·LBP complexes can induce little or no signal
response without the help of CD14
(10). Seydel and co-workers
(36) showed that LBP may
insert into monocyte membranes and enhance cell responses to LPS. In their
experiments, LBP did not inhibit monocyte activation when LBP and LPS were
added together, possibly because the LBP concentrations (
0.2 µg/ml) in
their experiments were below the threshold required for inhibition.
Although we used sCD14 to promote the binding of LPS to our cells, our data
suggest that there was no interaction between LBP and sCD14 in our
experiments. After the cells were washed to remove unbound LPS·sCD14
complexes, the cell-bound LPS cross-linked to mCD14 and not to sCD14. We
previously found that, under these conditions, sCD14 transferred LPS to mCD14
in an
1:1 molar ratio of LPS to mCD14
(34). Under these conditions,
all of the sCD14 is probably released from the cells, since the presence of
LBP is required to promote measurable cellular retention of sCD14
(37).
Other inhibitory mechanisms of LBP include its ability to form large
extracellular LPS-LBP complexes that have a reduced ability to stimulate cells
(30,
36,
38). As noted above, these
complexes appear to target most of the LPS for internalization. The ability of
LBP to enhance internalization of LPS may be restricted to LPS·LBP
complexes that are formed before LPS-cell binding occurs; we found that, once
LPS monomers were bound to mCD14, LBP had little or no ability to enhance LPS
internalization (Fig. 3). LBP
also inhibits LPS activity by transferring it to plasma lipoproteins, which
bind and neutralize LPS (6,
29). In a physiological
environment, such as plasma, it is likely that each of these mechanisms of
inhibition contributes to the cumulative inhibitory effect of LBP, but the
relative importance of each mechanism is difficult to determine.
Previous studies suggest that the balance between the stimulatory and
inhibitory effects of LBP depends primarily upon the LBP concentration. When
LBP and LPS were coincubated with macrophages in serum-free medium,
concentrations of LBP that are much lower than those found in normal plasma
(0.1 µg of LBP/ml) promoted maximal cell stimulation, whereas higher
concentrations (110 µg/ml) were less stimulatory, suggesting that
LBP was exerting a significant inhibitory effect at the higher concentrations
(27,
39). Whereas LBP can promote
LPS signaling at all LBP concentrations, the inhibitory mechanisms require
relatively high LBP concentrations to have a significant impact. In the same
experiments, high concentrations of LBP were inhibitory only when the LPS
concentration was low (
1 ng/ml), whereas at higher LPS concentrations,
much higher concentrations of LBP were required to inhibit responses to the
LPS (27,
28). LBP concentrations often
increase to very high levels in the blood of septic patients, and progressive
immunodepletion of LBP from the serum of these patients revealed that the LBP
can have a strong inhibitory effect on the activity of LPS
(28).
The balance between the stimulatory and inhibitory effects of sCD14 are
also concentration-dependent
(26), although the inhibitory
mechanism of sCD14 differs from that of LBP. Taken together, these findings
suggest mechanisms by which the moderate to high concentrations of LBP and
sCD14 that are found in human blood may help to prevent LPS-induced systemic
inflammation, whereas lower concentrations of these proteins, which presumably
occur in extravascular fluids, may promote beneficial inflammation at local
sites of infection
(4042).
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FOOTNOTES
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* This work was supported by National Institutes of Health (NIH) Grant
AI45896 from the NIAID, NIH grant HL 23584, State of California Grant TRDRP
11RT-0073, and NIH Grants PO1GM37696 and AI32021. The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
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To whom correspondence should be addressed: Dept. of Internal Medicine,
University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,
Dallas, TX 75390-9113. Tel.: 214-648-6479; Fax: 214-648-9478; E-mail:
richard.kitchens{at}UTSouthwestern.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; Tlr4, Toll-like
receptor 4; LBP, LPS-binding protein; mCD14, membrane-bound CD14; sSD14,
soluble CD14; sMD-2, soluble MD-2; VD3, 1,25-dihydroxyvitamin
D3; IL, interleukin; SFM, serum-free medium; SASD,
sulfosuccinimidyl-2-[p-azidosalicylamido]ethyl-13'-dithiopropionate;
ASD, 2-[p-azidosalicylamido]ethyl-1-3'-dithiopropionate;
PGE2, prostaglandin E2. 
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ACKNOWLEDGMENTS
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We thank Katrin Soldau for assistance with the production of rLBP and Dr.
Stephen F. Carroll of XOMA Corp. for generously providing the antibody to LBP.
We also thank Dr. Robert S. Munford for critically reading the manuscript.
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