(Received for publication, March 23, 1995; and in revised form, May 17, 1995)
From the
CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide
(LPS) and enables LPS-dependent responses in a variety of cells.
Monoclonal antibodies of CD14 such as 3C10 and MEM-18 are known to
neutralize biological activity of CD14. Recently, it has been
demonstrated that MEM-18 recognizes the LPS-binding site of CD14,
between amino acids 57 and 64. It has also been shown that 3C10
recognizes a distinct epitope from that of MEM-18, indicating that 3C10
may yet define another functional domain of CD14. In order to identify
the epitope for 3C10, we constructed a series of alanine substitution
mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions
between amino acids 7 and 10 and between amino acids 11 and 14 are
required for 3C10 binding. To assess the effect of altering the 3C10
epitope in CD14, we generated a stable cell line expressing a mutant
sCD14 containing alanine substitutions in the region between amino
acids 7 and 10, sCD14
CD14 is a 55-kDa glycoprotein that exists as a
glycosylphosphatidylinositol-anchored protein on the surface of
monocytes and neutrophils (PMN)
Several neutralizing mAbs to CD14 have been shown
to antagonize cellular responses to LPS in
vitro(1, 5, 9, 12, 13, 14, 15) and in vivo(16) . These mAbs may recognize functional
domains of CD14 important for its activity, and studies of the epitopes
of these mAbs, therefore, are essential in defining these domains. We
have demonstrated that mAbs MEM-18 and 3C10 recognize a sCD14 mutant
truncated at amino acid 152, indicating that epitopes for these two
mAbs are within the first 152 amino acids(8) . We further
characterized the epitope of MEM-18 and defined a region between amino
acid 57 and 64 that is essential for LPS binding(17) . Deletion
of this region not only disrupted binding of this mAb but also binding
of LPS.
The epitope for mAb 3C10 defines another functional domain
of CD14. This mAb appears to recognize a different region from that of
MEM-18 (8) , and binding of the mAb to sCD14 does not affect
LPS binding to sCD14(17) , suggesting that this epitope may be
involved in a cellular function other than LPS binding. In this report,
we identify the epitope of 3C10 by making a series of site-directed
alanine substitution mutants in sCD14. We show that the region between
amino acids 7 and 14 is required for 3C10 binding. We further
characterized this domain by generating a sCD14 mutant with alanine
substituted at amino acids 7-10 (sCD14
The ability of
sCD14
For examining the NF-
To map the epitope for mAb 3C10, a series of alanine
substitution mutants were generated by site-directed mutagenesis (Table 1). Plasmids containing cDNA sequences encoding different
sCD14 mutants were transfected into COS-7 cells, and CM from these
cells were examined for the expression of mutant sCD14 proteins by
Western blot (Table 1). With the exception of
sCD14
Figure 1:
BIAcore analysis of 3C10 binding to
alanine substitution mutants of sCD14. CM were collected from COS-7
cells transfected with no DNA (MOCK),
sCD14
Figure 2:
mAb 3C10 does not recognize purified
sCD14
Figure 3:
sCD14
Figure 4:
sCD14
Figure 5:
sCD14
Figure 6:
Inhibition of LPS-induced cellular
responses by sCD14
In this report, we mapped the epitope for neutralizing mAb
3C10 to the region between amino acids 7 and 14 of sCD14. Substitution
of alanine residues in this region prevented binding of 3C10 to sCD14.
These data are consistent with our previous finding (8, 17) that the 3C10 epitope is located within the
first 152 amino acids of sCD14 and is distinct from the epitope of
MEM-18 at residues 57-64. To help understand how the 3C10 epitope
contributes to CD14 function, we purified sCD14
The defect in sCD14
Since sCD14
We thank Viki Jacobsen for technical support.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, and purified this
protein to homogeneity. sCD14
has impaired
ability to mediate LPS-dependent IL-6 up-regulation in U373 cells,
integrin activation in neutrophils, and NF-
B activation in U373
cells. Purified sCD14
was, however, capable of
forming a stable complex with LPS in an LPS binding protein-facilitated
and LPS binding protein-independent fashion. The ability of
sCD14
to bind LPS was also demonstrated in
assays in which excess sCD14
inhibited
LPS-mediated tumor necrosis factor-
production in whole blood and
adhesion of polymorphonuclear leukocytes to fibrinogen. These data
strongly suggest that a region recognized by neutralizing monoclonal
antibody 3C10 contains a domain required for cellular signaling but not
for LPS binding.
(
)and as a
soluble protein found in
serum(1, 2, 3, 4) . CD14 binds one
molecule of lipopolysaccharide (LPS) in a reaction catalyzed by
LPS-binding protein (LBP)(5) , an acute phase serum
protein(6) . Complexes of LPS with CD14 have been shown to be
sufficient to elicit inflammatory responses in leukocytes (5, 7, 8) and endothelial
cells(9, 10, 11, 12) . Deletion
mutagenesis studies have demonstrated that the N-terminal 152 amino
acids of sCD14 are sufficient for mediating inflammatory responses
induced by LPS, suggesting that the LPS binding and cell signaling
domains of sCD14 are located within the first 152 amino
acids(8) .
).
This mutant was capable of binding LPS but was impaired in its ability
to mediate cellular responses to LPS.
Reagents
Recombinant soluble CD14 (rsCD14) and
recombinant LBP (rLBP) were constructed and purified as
described(5) . Concentrations of all purified proteins were
determined with a Micro BCA protein kit (Pierce) according to
manufacturer's specification. Since full-length rsCD14 terminates
at position 348 of the mature protein(5) , we herein refer it
as sCD14. The anti-CD14 mAb 3C10 was purified by
chromatography on Protein G from the conditioned medium (CM) of a cell
line from American Type Culture Collection (ATCC TIB 228). Rabbit
polyclonal anti-human CD14 antiserum was prepared as
described(8) . Rough LPS (Salmonella minnesota R60 or
Re595) and smooth LPS (Escherichia coli 0111:B4 or S.
minnesota wild-type) were purchased from LIST Biological
Laboratories (Campbell, CA). Enzymes for DNA manipulation were
purchased from Boehringer Mannheim.
Site-directed Mutagenesis
Nine alanine
substitution mutants of sCD14 were used in this study. Table 1summarizes the names and the amino acid residues
substituted in each mutant. The Transformer site-directed mutagenesis
kit (Clontech, Palo Alto, CA) was used as described previously (17) to generate cDNAs encoding alanine substitution mutants of
sCD14 cloned in a mammalian expression vector. The primers used for
each mutant are as follows:
5`-CGCCAGAACCTTGTGCAGCTGCCGCTGAAGATTTCCGCTGC-3` for
sCD14,
5`-GTGAGCTGGACGATGCAGCTGCCGCCTGCGTCTGCAACTTC-3` for
sCD14
,
5`-CCGCTGCGTCTGCGCAGCTGCCGCACCTCAGCCCGACTGG-3` for
sCD14
,
5`-GCAACTTCTCCGAAGCAGCTGCCGCCTGGTCCGAAGCCTTC-3` for
sCD14
,
5`-GAACCTCAGCCCGACGCAGCTGCAGCCTTCCAGTGTGTG-3` for
sCD14
,
5`-CCGACTGGTCCGAAGCAGCTGCGTGTGTGTCTGCAGTAGAG-3` for
sCD14
,
5`-CATGCCGGCGGTGCAGCTGCAGCGCCGTTTCTAAAGCGCG-3` for
sCD14
,
5`-GGTCTCAACCTAGAGGCAGCTGCAGCGCGCGTCGATGCGGAC-3` for
sCD14
, and
5`-GAGCCGTTTCTAAAGGCAGCTGCTGCGGACGCCGACCCG-3` for
sCD14
.
Transient Expression of Mutant sCD14 Proteins in COS-7
Cells
To express mutant sCD14 proteins, mammalian expression
vectors containing mutant sCD14 cDNAs were introduced into COS-7 (ATCC
CRL 1651) cells by electroporation. Conditions for electroporation and
generation of serum-free CM from transfected COS-7 cells were as
described(8) . Expression of mutant sCD14 was analyzed by
Western blot using anti-CD14 polyclonal antibody.
BIAcore Analyses of Interactions between sCD14 Mutants
and 3C10 mAb
Recognition of sCD14 mutant proteins by
neutralizing monoclonal antibody 3C10 was performed with a BIAcore
biosensor instrument. The instrument, CM5 sensor chips, and
amine-coupling kit were purchased from Pharmacia Biosensor. Briefly,
mAb 3C10 (200 µg/ml in 20 mM sodium acetate, pH 3.4) was
immobilized to a CM5 sensor chip by amine coupling according to the
manufacturer's specifications. The flow cell immobilized with
3C10 was then incubated in succession with solutions as detailed in the
following steps: step 1, COS-7 CM for 2 min, and step 2, HBS buffer (10
mMN-2-hydroxyethylpiperazine-N`-2-ethanesulfonic acid,
pH 7.5, 0.15 M NaCl, 3.4 mM EDTA, 0.005% (v/v)
surfactant P20 (Pharmacia Biosensor)) for 2 min. For regeneration, 10
mM HCl solution was injected for 2 min. Injection was
performed at a rate of 5 µl/min. To quantitate the binding of sCD14
mutants in COS-7 CM to immobilized 3C10, we calculated the relative
response unit (RRU). The RRU was obtained by subtracting the response
unit (RU) recorded just before injection of CM from the RU recorded
after injection of CM and a 2-min wash.
Purification of sCD14
The
expression vector containing the cDNA encoding sCD14 was stably transfected into Chinese hamster ovary cells deficient
in dihydrofolate reductase as described(5) . A single clone was
grown without serum to generate CM containing
sCD14
. Mutant protein was purified exactly as
described(17) , except immunoaffinity chromatography was
performed with anti-CD14 polyclonal antibody coupled to Sepharose 4B
(Pharmacia Biosensor). Purity of the sample was checked by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver
staining or Coomassie Blue staining. The changed amino acid sequence
was verified through N-terminal sequencing.
U373 Bioassays
Growth of U373 cells (ATCC HTB17,
Rockville, MD), activation by purified sCD14 preparations, and
quantitation of IL-6 were performed exactly as described(8) .
Briefly, mixtures of sCD14 or
sCD14
and LPS were added to monolayers of U373
cells in serum-free medium and incubated for 24 h. IL-6 in the
supernatant was then measured by enzyme-linked immunosorbent assay.
PMN Adhesion Assays
The ability of rLBP and
sCD14 or sCD14
to enable
PMN adhesion to fibrinogen-coated plates was assessed by previously
established protocols(5, 8) . Briefly, PMNs were
incubated for 10 min with LPS, rLBP, and sCD14
or sCD14
and washed, and adhesion to
fibrinogen-coated surfaces was measured as
described(5, 8) . When smooth LPS is used in this
protocol, adhesion is completely dependent on addition of
sCD14
(8) .
or sCD14
at high
concentrations to bind LPS and inhibit LPS-mediated PMN adhesion was
also assessed. In this experiment, rough LPS (S. minnesota R60, 10 ng/ml) was incubated with rLBP (1 µg/ml) and the
indicated concentrations of sCD14
or
sCD14
for 30 min at 37 °C before the addition
of PMNs. The adhesion of PMNs was measured as described above.
Electrophoretic Mobility Shift Assays
Whole cell
extracts from U373 cells were prepared to assess transcription factor
NF-B activation. Cells were seeded in 6-well plates at a density
of 1 million cells/well 1 day prior to stimulation. For stimulation,
purified sCD14
,
sCD14
(17) , or
sCD14
was added at a final concentration of 20
ng/ml with or without 20 ng/ml of Re595 LPS for 20 h. Cells were washed
twice with 1
phosphate-buffered saline (Life Technologies,
Inc.) and scraped in 200 µl of lysis buffer (20 mM HEPES,
pH 7.9, 20% glycerol, 0.1 M KCl, 1 mM EDTA, 0.5
mM dithiothreitol, 1 mM Pefabloc (Boehringer
Mannheim), 5 µg/ml leupeptin, 1 mM sodium orthovanadate,
and 2 µg/ml aprotinin) supplemented with 1% Triton X-100 (Sigma).
Crude extracts were transferred to microfuge tubes, and debris was
separated by centrifugation at 14,000
g for 10 min at
4 °C. Extracts were quickly frozen in liquid nitrogen and stored at
-80 °C. Protein concentration of the whole cell extracts was
determined by Micro BCA assay and ranged between 1.5 and 2
µg/µl.
B complexes, we performed
electrophoretic mobility shift assays. Two oligonucleotides
(5`-CATGGAGGGACTTTCCGCTGGGGACTTTCCAGC-3` and
5`-CATGGCTGGAAAGTCCCCAGCGGAAAGTCCCTC-3`) were annealed to generate a
double-stranded DNA containing the NF-
B binding site of human
immunodeficient virus long terminal repeat promoter(18) . This
annealed DNA fragment was then filled in with Klenow fragment
(Boehringer Mannheim) and [
-
P]dCTP
(Amersham Corp.) and used as probe at a concentration of 50,000
cpm/lane (about 25 fmol). For binding, 4 µl of whole cell extract
was incubated with 4 µl of 5
binding buffer (150 mM Tris-HCl, pH 8.0, 40 mM MgCl
, 5 mM
dithiothreitol, and 10% glycerol), 2.5 µg of
(poly(dI
dC)):(poly(dI
dC)) (Pharmacia Biosensor),
radioactively labeled DNA probe, and an adequate amount of lysis buffer
so that the final volume was 20 µl/reaction. The reactions were
incubated in a 30 °C water bath for 30 min, and complexes were
resolved in a native 4.5% polyacrylamide gel using 0.5
TBE (50
mM Tris-HCl, pH 8.0, 45 mM boric acid, and 5 mM EDTA) at 30 mA for 2 h. The gel was then vacuum-dried at 80 °C
for 1 h and exposed to Kodak x-ray film for 20 h. In competition
experiments, 100
molar excess of unlabeled NF-
B probe was
preincubated for 10 min before the addition of radioactive probe.
Native PAGE Assays
To directly assess LPS binding
of purified sCD14 preparations, sCD14 or
sCD14
was incubated at various concentrations
(0, 101, 303, and 909 nM) with 3 µg/ml of
H-LPS prepared from E. coli K12 strain LCD25 (List
Biological Laboratories) in the presence or absence of 16.7 nM rLBP. The reaction was incubated at 37 °C for 30 min and then
electrophoresed on native 4-20% polyacrylamide gels. Gels were
prepared for fluorography as described previously(5) .
Inhibition of LPS-Induced TNF-
The ability of sCD14 to bind LPS and inhibit TNF- Production in Whole
Blood
production in whole blood has been described(19) . Briefly,
various concentrations of bovine serum albumin (Miles, New Haven, CT),
sCD14
, or sCD14
diluted
in 50 µl of RPMI medium (Life Technologies, Inc.) were added to 250
µl of freshly drawn blood using heparin as an anti-coagulant.
Smooth LPS (S. minnesota wild-type) was added to a final
concentration of 0.25 ng/ml. The reaction was incubated at 37 °C
for 3 h, and supernatants were obtained by centrifugation at 16,000
g for 2 min. TNF-
concentrations in the
supernatants were assayed using a Quantikine TNF-
enzyme-linked
immunosorbent assay kit (R & D Systems, Minneapolis, MN) as
suggested by the manufacturer.
Alanine Substitution at Amino Acids 7-10 or
11-14 Disrupts Binding of Neutralizing mAb 3C10 to
CD14
3C10 is a mAb that recognizes the N-terminal 152 amino
acids of CD14(8) . Previous experiments have shown that 3C10
neutralizes the activity of
sCD14(1, 5, 9, 13) .
To verify that neutralization of sCD14 activity was due to binding of
epitopes within the N-terminal 152 amino acids, we demonstrated that
3C10 inhibited IL-6 production in U373 cells mediated by either
sCD14
or sCD14
(data not
shown).
, all sCD14 mutants were expressed and
secreted by COS-7 cells. BIAcore analysis (Fig. 1) was then used
to examine the ability of CM containing mutant sCD14 to bind 3C10. CM
containing sCD14
or sCD14
were found not to bind 3C10. These data suggest that the region
between amino acids 7 and 14 is involved in recognizing 3C10.
, or sCD14 mutants 4 days after
electroporation. All CM were analyzed for their ability to bind 3C10 as
described under ``Materials and Methods.'' RRUs were recorded
from four repeats of one experiment and calculated as means ±
standard deviations.
Purification and Characterization of
sCD14
Since neutralizing mAb 3C10
recognized amino acids 7-14, we reasoned that this region of CD14
could play an important role in the biological activity of CD14. To
help understand the role of this region, we generated a stable Chinese
hamster ovary cell line expressing sCD14 and
purified mutant protein from the serum-free CM of this cell line.
Purified sCD14
migrated with an apparent M
of 55,000 when analyzed by reducing SDS-PAGE
(data not shown). N-terminal sequencing indicated that the amino acids
between 7 and 10 were replaced with alanine residues as expected.
mAb 3C10 Does Not Recognize Purified
sCD14
BIAcore realtime analysis was
again used to determine whether mAb 3C10 is able to bind purified
sCD14. Fig. 2shows that
sCD14
recognized immobilized 3C10 and caused an
increase of 1800 RU 2 min after the wash (compare RU of the sensorgram
before HCl injection at t = 300 to that before
injection of sCD14
at t = 0),
confirming previous observations(8) . However, purified
sCD14
failed to recognize 3C10 and caused only
slight RU change (compare RU of the sensorgram after second wash at t = 750 to that before injection of
sCD14
at t = 0) similar to that
observed when an irrelevant protein such as bovine serum albumin was
injected (data not shown), demonstrating that amino acids 7-10
are required for mAb 3C10 binding.
. Immobilization of mAb 3C10 to a sensor
chip has been described(8) . 10 µg/ml sCD14
or sCD14
was used for injection.
Injections of solutions at various ``steps'' are marked on
the sensorgram. Wash indicates a washing step using
HBS buffer as described under ``Materials and Methods.'' The
experiments were performed three times, and the results of one
experiment are shown.
sCD14
To assess the
consequences of mutating residues between 7 and 10 in sCD14, we used
two previously described assays (5, 8, 9) to
measure sCD14Has Reduced Ability to
Mediate Cellular Responses to LPS
bioactivity. We first examined
the ability of sCD14
to enable responses of
U373 cells to LPS. Addition of as little as 5 ng/ml
sCD14
in the presence of LPS enabled strong IL-6
production (Fig. 3A). In contrast,
sCD14
was greatly impaired in its ability to
enable responses, and required approximately 10-fold more protein in
order to give a similar response to that of sCD14
(Fig. 3A).
is defective
in enabling cellular responses to LPS. A,
sCD14
has reduced ability to stimulate IL-6
production by U373 cells. U373 cells were treated with various
concentrations of sCD14
or
sCD14
in the presence or absence of LPS (20
ng/ml) for 24 h. IL-6 levels were determined as described(8) .
Data presented are means ± standard deviations from four
readings in an experiment repeated three times. B,
sCD14
but not sCD14
mediates responses of PMN to LPS and LBP. Freshly isolated PMN
were incubated with ``smooth'' LPS (E. coli 0111:B4,
30 ng/ml), rLBP (1 µg/ml), and the indicated concentrations of
sCD14
or sCD14
for 10 min
at 37 °C. Cells were washed, and adhesion to fibrinogen-coated
wells was measured(5, 25) . Errorbars indicate standard deviations of triplicate
determinations.
We also examined whether
sCD14 could enable LPS-induced adhesion of PMN
to fibrinogen. Fig. 3B shows that 100 ng/ml
sCD14
enabled a strong adhesive response of PMN
to smooth LPS and rLBP. However, very little response was seen even
when 10,000 ng/ml sCD14
was added. These
findings confirm that the region between amino acids 7 and 10 is
crucial for the biological activity of sCD14.
sCD14
LPS and sCD14-mediated activation of cells has been
shown to involve activation of transcription factors such as NF-Is Impaired in Its
Ability to Activate Transcription Factor NF-
B in the Presence of
LPS
B (20, 21, 22) . To assess whether the mutation
in sCD14
affected downstream signaling, we
examined NF-
B activation in U373 cells treated with wild type or
mutant sCD14. In the absence of LPS or sCD14, U373 cells possess
endogenous NF-
B, which forms a complex with labeled NF-
B
probe (Complex1; Fig. 4, lane1). Stimulation with LPS alone or sCD14
alone caused slight enhancement of NF-
B complex 1 and slight
induction of a new NF-
B complex (Complex 2, Fig. 4, lanes2 and 3), but addition
of sCD14
and LPS greatly induced both complexes
of NF-
B (Fig. 4, compare lanes1 and 4). Both complexes 1 and 2 are NF-
B-specific since a
100-fold excess of unlabeled NF-
B oligonucleotide preincubated
with extracts of U373 cells eliminated formation of both complexes
(data not shown). Stimulation of U373 cells with
sCD14
and LPS caused only 5% of NF-kB
activation as quantitated by gel scanning (Fig. 4, lane6). Comparatively, stimulation of U373 cells with a
mutant that does not bind LPS (sCD14
) failed
to activate NF-
B complexes even in the presence of LPS (Fig. 4, lane8). These data indicate that a
defect in sCD14
is observed at the level at the
transcription factor NF-
B. Since activation of NF-
B is an
early event in signal transduction(26) , these data suggest
that sCD14
fails to enable signaling.
does not
activate NF-
B. Whole cell extracts of U373 cells with various
treatments (control (lane1), LPS (lane2), sCD14
(lane3), sCD14
and LPS (lane4), sCD14
(lane5), sCD14
and LPS (lane6), sCD14
(lane7), and sCD14
and LPS (lane8)) were obtained, and binding of proteins to
the labeled NF-
B oligonucleotide was performed as described under
``Materials and Methods.'' Complexes of NF-
B were
resolved on a native 4.5% polyacrylamide gel. After electrophoresis,
the gel was dried and exposed to x-ray film for 16 h. Complexes of
labeled probe and NF-
B are indicated.
sCD14
Reduced signaling by sCD14Forms a Stable Complex
with LPS
could be due to a defect in binding LPS. To directly assess
whether sCD14
binds LPS normally, we used a
native PAGE assay to detect stable complexes between
sCD14
or sCD14
and
H-LPS. As previously reported(5) , formation of
stable complexes between sCD14
and LPS could be
observed after 30 min of incubation (Fig. 5A), and
addition of rLBP lowered the concentration of sCD14
required for complex formation (compare Fig. 5B, lane2, with Fig. 5A, lane2). This is consistent with the previous observation (5) that rLBP accelerates the transfer of LPS to sCD14.
Interestingly, sCD14
was also able to form
stable complexes with
H-LPS in the absence of rLBP (Fig. 5A, lanes5-7), and this
complex formation was also facilitated by rLBP (compare Fig. 5B, lane5, with Fig. 5A, lane5). These data confirm
that sCD14
is capable of binding LPS in an
LBP-facilitated and in an LBP-independent fashion in vitro and
suggest that the reduced biological activity of sCD14
is not due to an inability to bind LPS.
forms
stable complexes with
H-LPS. Various concentrations of
sCD14
(lanes2-4) or
sCD14
(lanes 5-7) were incubated
with 3 µg/ml
H-LPS in the absence (A) or
presence of 16.7 nM rLBP (B) as described under
``Materials and Methods.'' Lane1 contains
LPS in the absence of additional protein. Mixtures were run on
4-20% native polyacrylamide gels and processed for fluorography.
Positions of uncomplexed LPS and complexes between LPS and
sCD14
or sCD14
are
indicated.
Inhibition of LPS-induced Cellular Responses by High
Concentrations of sCD14
To further confirm that
sCD14 could bind LPS, we utilized two
cell-based assays in which high concentrations of sCD14 prevent
LPS-mediated activation of cells. In the first assay,
sCD14
or sCD14
was tested
for ability to inhibit adhesion of PMN to fibrinogen induced by LPS (Fig. 6A). In this experiment, constant concentrations
of LPS and rLBP were incubated with increasing amounts (from 1 to 100
µg/ml) of sCD14
or
sCD14
. Both proteins were capable of
neutralizing LPS and inhibiting the adhesion of PMN induced by LPS.
. A, inhibition of
LPS-induced PMN adhesion by sCD14
. Rough LPS (S. minnesota R60, 10 ng/ml) was incubated with LBP and
various concentrations of sCD14
or
sCD14
at 37 °C for 30 min before addition
of PMN. The adhesion of PMN to fibrinogen was measured as described
under ``Materials and Methods.'' Errorbars indicate standard deviations from three readings. B,
inhibition of TNF-
production in whole blood by
sCD14
. 250 µl of whole blood was incubated
with various concentrations of bovine serum albumin,
sCD14
, or sCD14
in the
presence of 0.25 ng/ml smooth LPS (S. minnesota wild type) at
37 °C for 3 h, and TNF-
production was measured as described
under ``Materials and Methods.'' Fraction of TNF
Production refers to the ratio of TNF produced in the presence of
exogenous protein divided by TNF produced in the absence of added
protein. Errorbars are standard deviations from six
readings.
We also examined whether sCD14 could inhibit
LPS-mediated TNF-
production in a whole blood assay, as has been
shown for a recombinant sCD14 expressed in Baculoviridae(19) .
Addition of increasing amounts of sCD14
or
sCD14
caused inhibition of TNF-
production
in the whole blood assay (Fig. 6B), while addition of
bovine serum albumin did not inhibit TNF-
production, confirming
the previous observation(19) . These data confirm that
sCD14
interacts with LPS as well as
sCD14
.
and showed that this protein was severely impaired in its ability
to activate cells. Inability of this protein to promote activation of
NF-
B suggests that sCD14
fails to support
LPS-mediated signaling.
signaling is unlikely to result from an inability of this protein
to bind LPS properly or to interact with LBP. sCD14
binds LPS normally, as examined by gelshift (Fig. 5A) and two cell-based assays (Fig. 6),
and rLBP facilitates transfer of LPS to sCD14
(Fig. 5B). These data confirm our previous
observation that 3C10 binds normally to complexes of sCD14 and
LPS(17) . We wish to point out that our experiments have
measured direct binding of LPS to sCD14
, not
the binding of LPS-LBP complexes to cell surface CD14 measured in other
reports(1, 23, 27) . Direct binding of LPS to
sCD14 is saturable with 1-2 LPS molecules bound per
CD14(5) , while binding of LPS
LBP complexes is not
saturable, with up to 1,000 LPS
LBP complexes bound to each CD14
at the cell surface(27) . Stoichiometric LPS-sCD14 complexes
stimulate cells, but the precise significance of the interaction of
multiple LPS
LBP complexes with CD14 is not clear at this time.
The above differences may explain our previous observation that mAb
3C10 blocked binding of erythrocytes coated with LPS
LBP complexes
to macrophages. The antibody may have sterically blocked the approach
of large LPS
LBP complexes to cell surface CD14. They may also
explain the recent observation of Viriyakosol and Kirkland (23) that deletion of amino acids 9-12 disrupted the
serum-dependent binding of LPS to CD14-bearing cells. This deletion may
have caused conformational changes in CD14 that disrupted direct
binding of LPS to CD14, binding of LPS-LBP complexes to CD14, or both.
The structural basis for binding of multiple LPS-LBP complexes to CD14
is not clear at this time, but it appears unlikely that a short
sequence of amino acids could support binding of 10-1,000
LPS
LBP complexes.
binds
LPS normally, its defect in signaling is likely to be manifest at the
cell membrane. We (9) and others (10, 11, 12) have postulated the existence of
a transmembrane protein that interacts with LPS and/or CD14 and
transmits signals to the cytoplasm. It is thus possible that residues
7-10 are essential for the interaction of sCD14 with this
transmembrane constituent. Alternatively, sCD14
may be defective in delivering LPS to the lipid bilayer of cells.
We have recently shown that sCD14 rapidly shuttles LPS into HDL
particles (24) and into phospholipid vesicles,
(
)and it is thus possible that residues 7-10 are
essential for delivery of bound LPS into the plasma membrane of cells.
Experiments are underway to distinguish these two possibilities.
B, nuclear
factor
B; PAGE, polyacrylamide gel electrophoresis; r,
recombinant; s, soluble; RRU, relative response unit; mAb, monoclonal
antibody; TNF, tumor necrosis factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.