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INTRODUCTION |
Endotoxic shock is an acute septic syndrome caused by the
overproduction of pro-inflammatory mediators during a bloodstream infection with Gram-negative bacteria. The pathogenesis of the shock is
presumed to be secondary to excessive stimulation of host cells by
bacterial lipopolysaccharide
(LPS,1 endotoxin), leading to
the synthesis and release of cytokines, arachidonic acid metabolites,
and various other mediators (1-3). The identification of CD14 as a
mediator of LPS-inducible signal transduction was a crucial event in
understanding the mechanism by which LPS-induced cellular activation
occurs. CD14, a 55-kDa glycosylphosphatidylinositol (GPI)-linked
protein present on the surface of phagocytic leukocytes, has been shown
to bind LPS and to mediate cellular activation (4-6). In addition, a
soluble form of CD14 (sCD14) is also capable of binding LPS and
activating some CD14-deficient cells, such as endothelial cells
(7-10).
Although it is generally agreed that the interaction between lipid A
and CD14 is central to cellular activation by LPS, details of the
downstream events, which follow, remain obscure. For example, because
CD14 lacks a transmembrane domain, it is unlikely that CD14 alone is
responsible for directly transmitting a signal across the plasma
membrane. Many groups have hypothesized that CD14 and LPS must
interact, either directly or indirectly, with a second transmembrane
receptor, which would be the actual LPS signal transducer (reviewed in
Ref. 11). Alternatively, CD14 could mediate cellular activation via a
biophysical mechanism. Wright and colleagues (12, 13) observed that
CD14 could move LPS into lipid bilayers that resemble mammalian cell
membranes. Furthermore, they observed that trafficking of LPS from the
membrane to the Golgi correlated with the ability to induce signaling.
The CD11/CD18 (
2) integrins are a second group of LPS
receptors (14). The observation that CD11/CD18 could enable LPS
responsiveness independently of CD14 when expressed on the surface of
CHO cells (15, 16) suggested that the ability of CD14 to participate in
the LPS signaling cascade was not unique. We hypothesized that the
common function shared by these two LPS receptors was their ability to
bind LPS and bring it in close proximity to the plasma membrane, where
it could then interact with a signal transducer. To further investigate
the role of LPS binding in cellular signaling, we decided to exploit
the activity of two other proteins known to bind LPS,
lipopolysaccharide-binding protein (LBP) and
bactericidal/permeability-increasing protein (BPI).
LBP and BPI are members of the lipid transfer/lipopolysaccharide
binding gene family, which includes cholesteryl ester transfer protein
and phospholipid transfer protein (reviewed in Ref. 17). LBP and BPI
share approximately 45% homology at the amino acid level and are
encoded in the same region of human chromosome 20, suggesting that they
arose by gene duplication (18, 19). However, the two proteins appear to
have quite different biological functions. LBP, a soluble protein
secreted by hepatocytes into the blood stream, accelerates the binding
of LPS monomers to CD14 (20, 21). Thus, it enhances the sensitivity of
cells to LPS, and may be important in the host recognition of and
response to Gram-negative bacteria (11). In contrast, BPI, which is
found in the azurophilic granules of neutrophils, is bactericidal
toward Gram-negative bacteria, and inhibits the biologic activity of
LPS (22, 23).
To test the hypothesis that cellular binding alone of LPS would be
sufficient to initiate cellular signaling, we constructed chimeric
proteins consisting of LBP or BPI attached to the GPI anchor of decay
accelerating factor (DAF) (24, 25). Expression of these constructs
would thus create an artificial LPS receptor on the surface of the
transfected cell. We found that CHO-K1 fibroblasts expressing
GPI-anchored LBP or BPI could bind both LPS and Gram-negative bacteria.
Furthermore, they could initiate the LPS signaling cascade in a
CD14-independent manner, similar to CD11/CD18-transfected cell lines.
We conclude from this that the ability of an LPS binding protein to
focus LPS on the surface membrane of a cell, where it can interact with
a second receptor, is sufficient to activate the specific LPS-signaling apparatus.
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EXPERIMENTAL PROCEDURES |
Reagents--
PBS, Ham's F-12, and Trypsin-Versene mixture were
obtained from Bio-Whittaker (Walkersville, MD). Ex-Cell 301 serum-free
medium was obtained from J.R.H. Biosciences (Lenexa, KS), fetal calf serum (FCS) (LPS less than 10 pg/ml) from Summit Biotechnology (Greeley, CO), G418 from Life Technologies, Inc., ciprofloxacin from
Miles Pharmaceuticals (West Haven, CT), and trypan blue solution (0.4%) from Sigma. Human LBP and soluble CD14 were gifts of Henry Lichenstein (Amgen, Boulder, CO). Purified monoclonal antibody 3C10 was
a gift of Terje Espevik and Egil Lien (Norweigan University of Science
and Technology, Trondheim, Norway). LPS from Salmonella minnesota R595 (ReLPS) was a gift from Nilo Qureshi and Kuni
Takayama (University of Wisconsin, Madison, WI). LPS was protein-free
by Bio-Rad protein assay (Bio-Rad). Compound B287 (26) and compound B1287 (patent reference number WO-9639411-A1) were prepared at Eisai
Research Institute (Andover, MA). Lipids were prepared as 1 mg/ml
dispersed sonicates in pyrogen-free PBS and stored at
20 °C.
Before use, the suspensions were thawed and sonicated for 3 min in a
water bath sonicator (Laboratory Supplies, Hicksville, NY) before
diluting to final concentration.
Construction of DAF Fusion Proteins--
All fusion proteins
were cloned by PCR using Pfu (Stratagene, La Jolla, CA). The
3'-end of DAF, containing the GPI anchor, was obtained from the
cDNA of vitamin D3-treated THP-1 cells using PCR
primers (5'-primer GCATGATATCTTGAAACAACCCCAAATAAAGGAAGTG, 3'-primer GCTCTAGATCTCAATTCTGCAAGTTGTTCTATTTTCA) under the following conditions: (94° 1:00, 54° 0:45, 72° 2:00) × 40 cycles;
(72° 5:00, 35° 5:00) × 1 cycle. This DAF fragment was inserted
into pCDNA3 (Invitrogen, San Diego, CA); the cDNAs for LBP,
BPI, and CD4 were then subsequently inserted into this vector 5' to the
DAF GPI fragment.
Human LBP was cloned from a plasmid containing human LBP cDNA in
the pCMV vector (gift of Ralf Schumann, Molecular Sepsis Research
Laboratory, Berlin, Germany). The PCR primers (5' primer GAAGAATTCATGGGGGCTTTGGCA, 3' primer CCCTGTACACGCATGTATTGGACA) were used
under the following conditions: (94° 1:00, 50° 0:45, 72° 2:30) × 5 cycles; (94° 1:00, 55° 0:45, 72° 2:30) × 5 cycles; (94°
1:00, 60° 0:45, 72° 2:30) × 20 cycles; (72° 5:00, 28° 5:00) × 1 cycle.
Human BPI was cloned from a human bone marrow cDNA library
(CLONTECH, Palo Alto, CA). The PCR primers (5'
primer GGCAGTTCGGTACCATGAGCGAGAACAT, 3' primer
TGGTGCCAGCGCTTATAGACAACG) were used under the following conditions:
(94° 1:00, 52° 1:00, 72° 2:30) × 5 cycles; (94° 0:30, 57°
1:00, 72° 2:30) × 15 cycles; (72° 5:00, 28° 5:00) × 1 cycle.
Human CD4 was cloned from a plasmid containing human CD4 cDNA in
the CDM8 vector (gift of Ellis Reinherz, Ref. 27). The PCR primers (5'
primer CAAGGAATTCATGAACCGGGGAGTC, 3' primer CACGATCATGCGCATTGGCTG) were
used under the following conditions (94° 1:00, 52° 1:00, 72°
2:30) × 5 cycles; (94° 0:30, 57° 1:00, 72° 2:30) × 15 cycles; (72° 5:00, 28° 5:00) × 1 cycle.
All three chimeric proteins have two additional amino acids at the site
of fusion, as shown in Fig. 1. The in-frame fusion of all constructs
was confirmed by DNA sequencing using the dideoxy chain termination
method (28). In addition, the LBP and BPI portions were sequenced in entirety.
Cell Lines--
Chinese hamster ovary fibroblast (CHO-K1) cells
were obtained from the American Type Culture Collection (Manassas, VA)
and maintained in Ham's F-12 supplemented with 10% FCS and 10 µg of ciprofloxacin/ml (complete medium). Cell lines were grown as adherent monolayers in tissue culture dishes at 37 °C in 5% CO2,
and passaged twice a week to maintain logarithmic growth.
The following stably transfected cell lines have been described:
CHO/Neo, CHO-K1 transfected with the pCDNA1/Neo vector (29); and,
CHO/CD14, human CD14 transfected CHO-K1 (29). CHO/CD4, CHO/LBP, and
CHO/BPI were engineered by stably transfecting CHO-K1 with the DAF
constructs by calcium phosphate precipitation (29). Stable
transfectants were selected in medium supplemented with G418 (1 mg
active drug/ml), and surface expression of the transfected genes was
confirmed by flow cytometry using a FACScan flow cytometer (Becton
Dickinson). Bulk transfected cells were subjected to one round of
positive selection using a Becton Dickinson FACScan Plus fluorescence
activated cell sorter in enrichment mode to select for cells with the
highest levels of the transfected receptor. A clonal cell line was then
selected from this population by limiting dilution cloning.
The following antibodies were used for flow cytometry: goat anti-human
LBP antibody (1:100 dilution; gift of Ralf Schumann, Molecular Sepsis
Research Laboratory, Berlin, Germany) and fluorescein isothiocyanate-conjugated anti-goat IgG (1:100 dilution; Sigma); rabbit
anti-human BPI (1:100 dilution; gift of Russell Dedrick, XOMA Corp.,
Berkley, CA) and fluorescein isothiocyanate-conjugated anti-rabbit IgG
(1:100 dilution; Sigma); fluorescein isothiocyanate-conjugated anti-CD4
antibody (Leu-3a; 2:5 dilution; Becton Dickinson).
Cell Culture and Stimulation Conditions--
One day before
stimulation, cells were plated in 6-well tissue culture dishes at a
density of 5 × 105/well. On the day of stimulation,
wells were aspirated and washed three times with PBS to remove FCS, and
medium was replaced with 1 ml of Ham's F-12 with 2% FCS or Ex-Cell
serum-free medium. When LBP and soluble CD14 were used in assays, they
were added to the Ex-Cell medium for a final concentration of 200 ng/ml
or 100 ng/ml, respectively. For antibody experiments, cells were
preincubated with 3C10 (20 ng/ml) on ice for 20 min before the addition
of LPS.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
After stimulation, cells were washed with PBS,
2% FCS, harvested using a rubber policeman, and pelleted in a
microcentrifuge (Beckman Microfuge 11). Nuclear proteins were isolated
as described in detail previously (30). The extracted proteins were
then assayed for the presence of nuclear factor-
B (NF-
B) as
described using a 32P-labeled oligonucleotide containing
the consensus sequence for NF-
B binding from the murine
immunoglobulin
light chain gene enhancer. The DNA-protein binding
reactions were analyzed by nondenaturing gel electrophoresis. Gels were
transferred to filter paper, dried, and exposed to x-ray film (30).
LPS Binding and Phagocytosis Assays--
LPS binding assays with
boron dipyrromethane (BODIPY)-labeled LPS were performed as described
(31) except that the assay was adapted for whole cell binding.
Complexes of BODIPY-LPS (100 ng/ml; gift of Rolf Thieringer, Merck
Research Laboratories) and soluble CD14 (2 µg/ml) were preformed
overnight at 37 °C. CHO-K1 transfectants were plated at a density of
2 × 105/well in a 1-ml volume, and allowed to grow
overnight. BODIPY-LPS/sCD14 complexes were then added to adherent
monolayers in the presence or absence of recombinant human
lipopolysaccharide binding protein (200 ng/ml) for 30 min at 37 °C.
Following the incubation period, wells were washed with PBS containing
1% feral bovine serum, and trypan blue (0.4%) was added for a 1-min
incubation period to quench extracellular fluorescence. Cells were
detached using trypsin-versene, and analyzed for fluorescence using
flow cytometry (29).
For whole bacteria phagocytosis assays, cells were treated as above
except that BODIPY-conjugated Escherichia coli (K-12 strain) BioParticles (Molecular Probes, Eugene, OR) were added to the CHO-K1
monolayers to give a final concentration of 3 × 107/ml. Binding and internalization of the BioParticles by
the CHO cells were allowed to proceed for 1 h. Before analysis,
fluorescent extracellular bacteria were quenched using trypan blue.
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RESULTS |
CHO-K1 Cells Expressing LBP or BPI Bind LPS and Gram-negative
Bacteria--
Shown in Fig. 1 are the
DNA and amino acid sequences of LBP, BPI, and CD4 at the site of fusion
with DAF. CHO-K1 fibroblasts were stably transfected with the three DAF
plasmids to create the two experimental cell lines, CHO/LBP and
CHO/BPI, and the control cell line, CHO/CD4. Surface expression of the
chimeric proteins in clonal cell lines was confirmed by flow cytometry (Fig. 2). Whereas FACS is
semi-quantitative, relative protein expression between cell lines could
not be calculated from this analysis, because each cell line was
stained using different primary and secondary antibodies. All cell
lines, however, did express significant immunofluorescent signals
compared with the isotype-stained control cells.

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Fig. 1.
Construction of CD4-DAF, LBP-DAF, and
BPI-DAF. CD4-DAF, LBP-DAF, and BPI-DAF were constructed as
described in the text. Shown above are the DNA and amino acid sequences
of the chimeric proteins at the site of fusion. The 3' sequences of
CD4, LBP, and BPI are shown on the left, whereas the 5' end of the GPI
anchor from DAF is on the right. Each chimeric protein has two
additional amino acids (shown in bold) at the site of fusion
created by the ligation of the DNAs.
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Fig. 2.
Surface expression of chimeric proteins in
the CHO transfectants. Transfected cell lines were subjected to
flow cytometry analysis using the antibodies described in the text. A
fluorescence-activated cell sorter histogram of CHO/CD4, CHO/LBP, and
CHO/BPI is shown above. Fluorescence intensity is graphed on the
x axis, whereas cell number is on the y
axis.
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The ability of LBP and BPI to bind LPS as soluble proteins is well
described (32, 33). We wanted to be certain that surface-expression of
these proteins did not interfere with this function. To assess the
ability of the transfectants to bind LPS, CHO/LBP, CHO/BPI, CHO/CD4 and
CHO/CD14 were incubated with preformed complexes of BODIPY-LPS and
soluble CD14, an experimental technique that is thought to dissociate
LPS aggregates into monomers (31). Cells were then analyzed by flow
cytometry for fluorescence. Only the cell lines expressing the
LPS-binding proteins (LBP, BPI, and CD14) bound BODIPY-LPS (Fig.
3). Co-incubation of soluble LBP had no
effect on the observed binding of LPS-sCD14 complexes to the CHO/LBP
and CHO/BPI lines. In contrast, binding to CHO/CD14 was greatly
enhanced when LPS-sCD14 complexes were added in the presence of soluble
LBP, consistent with previous observations that LBP specifically
promotes movement of LPS onto CD14 (31).

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Fig. 3.
Binding of LPS in the CHO transfectants.
Binding assays were carried out as described in the text. BODIPY-LPS
was added as a preformed complex with soluble CD14, either in the
presence or absence of LBP. Cells were analyzed for fluorescence by
flow cytometry analysis. The histograms for each cell line are shown
above. On the x axis is graphed fluorescence in the FL-1
channel, whereas the y axis represents the relative number
of cells. For comparison to "background" LPS binding, the graph for
the CHO/CD4 control line is reproduced in the remaining
histograms.
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Previous reports by Tobias and colleagues (34) demonstrated that the
affinity of BPI for LPS was higher than that for LBP. However, our data
suggests that binding to the CHO/LBP line is better than that of
CHO/BPI. In part, this may be due to changes in the conformation of the
protein in its anchored state. In addition, Tobias' data was obtained
through a careful titration of the molar ratios of LPS to the binding
proteins, something which would be more difficult to accomplish in our
cell lines, because we cannot control for the exact level of surface
expression of LBP, BPI and CD14. However, the data does demonstrate, at
least in a qualitative manner, that LBP and BPI function as effective
endotoxin-binding proteins when expressed as chimeric GPI-linked
proteins on the surface of CHO cells.
Other groups (35, 36) have described the ability of CD14 to enable the
internalization of Gram-negative bacteria, presumably through its
interaction with a transmembrane receptor. We wanted to know whether
the GPI-anchored LBP and BPI were also capable of enhancing bacterial
uptake. When CHO/LBP, CHO/BPI, CHO/CD4 and CHO/CD14 were incubated with
fluorescent E. coli BioParticles, we found, again, that only
the lines expressing the LPS-binding proteins were capable of binding
and internalizing the bacteria (data not shown).
CHO-K1 Cells Expressing LBP or BPI Respond to LPS--
To assess
the ability of the membrane anchored LBP and BPI to mediate LPS
activation, cells were incubated with increasing doses of LPS, and
nuclear extracts were analyzed for the induced translocation of
NF-
B. We found that expression of LBP or BPI enabled CHO-K1 to
respond to LPS in both the absence and presence of serum (Fig.
4). The response to LPS in the control
line, CHO/CD4, was negligible under both conditions. When the
individual lines were compared, we found that CHO/BPI was approximately
10-fold more sensitive to LPS relative to CHO/LBP. Both cell lines were less sensitive to LPS when compared with CHO/CD14. The
LPS-responsiveness was not felt to be unique to the individual clones
studied, because a nonclonal population of LBP-expressing cells,
derived following one round of FACScan sorting, responded
similarly.

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Fig. 4.
LPS activates the CHO/LBP and CHO/BPI
transfectants. Clonal cell lines derived from the CHO
transfectants CHO/CD14, CHO/LBP, CHO/BPI, and CHO/CD4 were treated with
increasing doses of LPS under serum-free conditions. Nuclear proteins
were prepared, and nuclear levels of NF- B were measured by EMSA
using a B site-containing probe. Only the band representing NF- B
bound to the B site-containing probe is shown. Similar results were
found in the presence of 2% FCS.
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The time course of NF-
B translocation also differed between the
CD14, LBP, and BPI lines. The response to LPS in CHO/CD14 is rapid; it
begins within 15-30 min, peaks at 1 h, and rapidly returns to
baseline by 3 h. For the CHO/LBP and CHO/BPI lines, however, the
signaling peaked between 30 min and 1 h, and was sustained even
after 3 h (Fig. 5). This is similar
to the time course observed previously with the CD11/CD18 expressing
CHO cells (15, 16). The more transient activation seen with CD14 in comparison with the other receptors suggests that it alone may activate
a divergent pathway involved in down-regulation of the signal.

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Fig. 5.
Time course of NF- B
activation in CHO transfectants. CHO/CD14, CHO/LBP, CHO/BPI, and
CHO/CD4 were stimulated with LPS (100 ng/ml) for increasing time
periods. Nuclear proteins were prepared, and levels of NF- B were
measured by EMSA.
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LPS Signaling in CHO/LBP and CHO/BPI Is Independent of Soluble CD14
and Soluble LBP--
Despite the use of strict serum-free conditions,
we wanted to be certain that the signaling we observed in CHO/LBP and
CHO/BPI was not being mediated through soluble CD14 contaminating our cell culture system. Historical evidence suggested that this was not
the case. First, CHO cells do not express endogenous CD14 mRNA by
Northern blot analysis, making it unlikely that they would produce and
secrete the protein into the supernatant (29). In addition, unlike
endothelial cells and several other non-CD14 expressing cell types,
neither soluble CD14 nor serum has been shown to enable wild-type or
mock transfected CHO-K1 cells to respond to low doses of LPS (29,
30).
Further evidence came from the use of 3C10, an anti-CD14 monoclonal
antibody which has been shown to specifically inhibit LPS signaling
(5). We found that 3C10 inhibited LPS signaling in CHO/CD14, whereas
the response to LPS by CHO/LBP and CHO/BPI was not diminished in the
presence of this antibody (Fig. 6). Finally, we found that the addition of soluble LBP or soluble CD14 to
the culture medium did not shift the sensitivity of the CHO/LBP line to
LPS. This was in contrast to what has been described with CD14-mediated
signaling, further demonstrating that the activity that we observed was
independent of CD14 (Fig. 7).

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Fig. 6.
Effect of anti-CD14 antibody on activation in
CHO/LBP and CHO/BPI. CHO/CD14, CHO/LBP, and CHO/BPI were prepared
and plated under serum-free conditions. Cells were preincubated with
3C10 before stimulation with varying concentrations of LPS for 1 h. Nuclear extracts were prepared, and levels of NF- B were measured
by EMSA.
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Fig. 7.
LPS activation of CHO/LBP does not require
soluble CD14 or LBP. CHO/LBP and CHO/Neo were treated with
increasing doses of LPS in the presence or absence of recombinant human
LBP (200 ng/ml) or soluble CD14 (100 ng/ml). Nuclear proteins were
prepared, and levels of NF- B were measured by EMSA.
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The Effects of Lipid A Analogues in CHO/LBP and CHO/BPI Are
Identical to That of CHO/CD14--
Pharmacologic studies with
biologically derived lipid A antagonists have led to the hypothesis
that CD14 activates cells, not directly, but via an ancillary signaling
molecule. The first line of data comes from careful binding studies by
Kitchens and colleagues (37, 38) demonstrating that these compounds
inhibit the ability of LPS to activate cells at concentrations that are too low to inhibit binding of LPS to CD14. In addition, the
pharmacological effects of the antagonists suggest a complex signal
transduction apparatus for LPS. For example, Rhodobacter
sphaeroides lipid A (RSLA) and lipid IVA are potent
LPS antagonists in LPS-responsive human cells (39-41), whereas in
hamster and mouse cells these compounds have very different effects. In
hamsters, both lipid IVA and RSLA are LPS mimetics (42). In
mice, lipid IVA is an LPS mimetic and RSLA is an LPS
antagonist (39, 40, 43, 44). Data from transfected cell lines has shown
that the species-specific effects of these compounds are determined not
by the species of CD14, but by the genome of the host cell on which it
is expressed (42). Taken together, these data suggest that the
inhibitors are antagonizing LPS at a site distinct from CD14.
The first compound we tested was B287, a synthetic lipid A analogue
based on the proposed structure of RSLA. B287 has activity identical to
that of natural RSLA when tested in macrophage cell lines and whole
human blood ex vivo (26), and has been previously shown by
our laboratory to be a potent LPS mimetic in CHO/CD14 cells (42). When
CHO/LBP was incubated with increasing doses of B287 we found that the
nuclear translocation of NF-
B was induced (Fig.
8). Similar results were found with
CHO/BPI (data not shown). Thus the compound acted as an LPS mimetic in
CHO/CD14, CHO/LBP, and CHO/BPI. Use of this synthetic LPS mimetic was
also strong evidence that the cellular activation we observed with our
ReLPS was not occurring via any contaminating bacterial proteins in the
preparation.

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Fig. 8.
The synthetic LPS analogue, compound B287,
activates CHO/LBP, whereas compound B1287 inhibits LPS signaling.
CHO/LBP was treated with increasing doses of LPS, compound B287,
compound B1287, or LPS with B1287. Nuclear proteins were prepared, and
nuclear levels of NF- B were measured by EMSA using a B
site-containing probe. The bands represent DNA probe bound to nuclear
NF- B. Similar results were found with CHO/BPI (data not
shown).
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The next compound we examined was B1287, which we recently described as
a potent antagonist in the LPS responsive CHO/CD14 cell line (45). When
this compound was tested in the CHO/LBP line we found a similar result;
it had no intrinsic LPS-like activity and was capable of completely
blocking the LPS-induced cellular activation in our assay (Fig. 8).
Similar results were found for CHO/BPI (data not shown). Thus, the
effects of B287 and B1287 are the same in CHO/CD14, CHO/LBP, and
CHO/BPI.
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DISCUSSION |
The current understanding of LPS signaling consists of LPS binding
to either membrane or soluble CD14 followed by cellular activation. The
mechanism by which this occurs, at least at the molecular level, is
still unclear. The central role of CD14 in the process is supported, in
part, by the observation that CD14-deficient mice are highly resistant
to the pro-inflammatory effects of LPS and Gram-negative bacteria (46).
However, although CD14 is clearly an important LPS binding protein that
participates in the initiation of LPS signaling events, one could argue
that it is not truly an LPS receptor in the sense that it does not
directly activate the signaling cascade. Furthermore, this ability to
participate in LPS signaling events is not a characteristic that is
unique to CD14. In fact, we have shown that other known surface
LPS binding proteins, specifically the CD11/CD18 (
2)
integrins, are also sufficient for imparting LPS responsiveness
when overexpressed in CHO-K1 cells (15, 16, 47).
As soluble LPS-binding proteins, LBP and BPI have never been shown to
enable LPS signaling in the absence of CD14. In fact, BPI has been
shown to inhibit LPS activity (22, 23). Thus, the ability of a soluble
LPS-binding protein to participate in cell activation appears to be
unique to soluble CD14. In contrast, our data demonstrates that LBP and
BPI, when expressed on the surface of a CHO cell, can enable cellular
activation in a manner similar to that of membrane CD14. One possible
interpretation of the data is that high expression of LBP on the
surface of the CHO cell enhanced signaling by efficiently transferring
LPS to any contaminating soluble CD14 in our system. However, this
interpretation would not explain the signaling observed in the CHO/BPI
line, because BPI does not possess the same LPS-transferase activity as
that of LBP (48).
Both LBP and BPI share the ability to bind LPS with high affinity, and
we believe that it is their ability to bind LPS and bring it in close
proximity to the cell surface that enables them to mediate activation
of the LPS signaling cascade. We believe that LPS is activating the
CHO/LBP and CHO/BPI lines in the same manner as CHO/CD14. This is
supported by the ability of the specific LPS antagonist B1287 to block
signaling events. However, there appears to be at least one unique
aspect of CD14-mediated signaling, at least in the transfected CHO
lines
the time course of cellular activation. Unlike LBP, BPI, and
CD11/CD18, CD14 is the only LPS-binding protein that turns off the
signal for NF-
B translocation after 1 h; the other three lines
all consistently have been shown to sustain the signal for more than
3 h. In addition, CHO cells co-transfected with both CD14 and
CD11/CD18 show a time course consistent with activation via both
receptors with a rapid peak and a sustained activation for up to 5 h.2 Thus, expression of these
surface LPS binding proteins in our system has not entirely duplicated
CD14-initiated signaling. It is possible that the signaling events
initiated by CD14 diverge from those of the other LPS receptors at some
point on the way to NF-
B translocation. In fact, this ability of
CD14 to down-regulate its own signal may hold physiologic relevance.
These data are consistent with our current model of LPS signaling in
which CD14 binds LPS and brings it in close proximity to the cell
membrane where it can then interact with a transmembrane receptor. This
second protein would be the actual LPS signal transducer/receptor. Alternate models of LPS signaling, whereby LPS uptake or intercalation into the membrane is an integral part of the downstream signaling events, are supported less well. Although LBP and CD14 can transport LPS into phospholipid bilayers (49), neither BPI nor the CD11/CD18 integrins are known to have such activity. The important observation appears to be that any protein that can bind LPS and concentrate it on
the cell membrane is capable of activating the LPS-specific signaling
pathway. These LPS-binding proteins thus enhance sensitivity to
endotoxin, but lack the specificity usually observed with true signal
transducing receptors.
Recently two Toll-like receptors, TLR-2 and TLR-4, have been implicated
in LPS signaling (50-53), and it is possible that one of the TLRs
receive endotoxin from these natural and engineered surface receptors.
However, any conclusions about the relevance of the TLRs in LPS
signaling will have to account for the properties of the anchored
LPS-binding proteins, as well as the species-specific effects of the
LPS inhibitors. The ability of GPI-anchored LBP and BPI to enhance
signaling in response to LPS exposure underscores the complex
interactions between the multiple receptors involved in LPS recognition
and cellular activation.