From the
Like other tetraacyl partial structures of lipopolysaccharide
(LPS) and lipid A, LPS that has been partially deacylated by
acyloxyacyl hydrolase can inhibit LPS-induced responses in human cells.
To identify the site(s) of inhibition in the LPS recognition pathway,
we analyzed the apparent binding affinities and interactions of
Lipopolysaccharide (LPS
The basis for the LPS-specific inhibitory action shown
by these analogs is controversial. As initially suggested by Pohlman
et al. (4) , the analogs may compete with LPS for
binding certain critical molecules in the LPS recognition pathway. A
likely target would be CD14, an important LPS receptor that, in the
presence of LPS binding protein (LBP), mediates cellular responses to
low concentrations of LPS
(13, 14, 15, 16) . In keeping with this
notion, other investigators
(17, 18, 19, 20) have found that high concentrations of synthetic tetraacyl
lipid A analogs ( e.g. LA-14-PP and PE-4) can inhibit LPS
binding to monocytes, presumably by competing with LPS for binding
CD14. In contrast, we found that dLPS and LA-14-PP can inhibit
responses to LPS without inhibiting CD14-mediated LPS uptake by cells
of the human THP-1 monocytic cell line
(8) . To attempt to
resolve this issue, we performed quantitative analyses of the binding
of [
A quantitative dissection of potential inhibitory mechanisms
has not been reported previously for any of the lipid A analogs that
are known to act as LPS antagonists. Two measurements were critical for
such an analysis. We first compared the binding affinity of the
antagonistic LPS analog (dLPS) to that of LPS. Competition between LPS
and dLPS for binding CD14 would be expected to occur at or near their
respective binding K
We found
that dLPS (and presumably other lipid A analogs) can block LPS
signaling in at least three ways as follows.
There are several
models that could explain the third mechanism of dLPS inhibition. The
idea that dLPS might block the binding of LPS to a high affinity
subclass of CD14 cannot be formally excluded. Under certain conditions,
we have observed high affinity binding of LPS to CD14, with an apparent
dissociation constant ( K
A more
likely model, shown in Fig. 7, postulates the existence of a low
abundance receptor/effector molecule that binds to LPS or dLPS either
in the context of CD14 (LPS
[
Low
concentrations of dLPS inhibit LPS binding to CD14 when LBP is
limiting. VD3-induced THP-1 cells (4
We thank Leon Eidels for critical review of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H-labeled enzymatically deacylated LPS (dLPS) and
[
H]LPS with CD14, the LPS receptor, on THP-1
cells. Using (i) incubation conditions that prevented ligand
internalization and (ii) defined concentrations of LPS binding protein
(LBP), which facilitates LPS and dLPS binding to CD14, we found that
dLPS can antagonize LPS in at least three ways. 1) When the
concentration of LBP in the medium was suboptimal for promoting
LPS-CD14 binding, low concentrations of dLPS were able to compete with
LPS for binding CD14, suggesting competition between LPS and dLPS for
engaging LBP. 2) When LBP was present in excess, dLPS could compete
with LPS for binding CD14, but only at dLPS concentrations that were at
or above its K
for binding CD14 (100
ng/ml). 3) In contrast, substoichiometric concentrations of dLPS (1
ng/ml) inhibited LPS-induced (3 ng/ml) interleukin-8 release without
blocking LPS binding to CD14. Functional antagonism was possible
without competition for cell-surface binding because both LPS-induced
interleukin-8 release and dLPS inhibition occurred at concentrations
that were far below their respective CD14 binding K
values. In addition to its expected ability to compete with LPS
for binding LBP and CD14, dLPS thus potently antagonizes LPS at an
undiscovered site that is distal to LPS-CD14 binding in the LPS
recognition pathway.
(
)
or endotoxin) is
a bioactive glycolipid constituent of the outer membranes of
Gram-negative bacteria. Responses to LPS in human cells show stringent
requirements for certain structural features of lipid A, the bioactive
center of LPS, including a diglucosamine backbone that bears both 1 and
4` phosphates and acyloxyacyl-linked (secondary) fatty acids
(1) . For example, a synthetic tetraacyl lipid A partial
structure that lacks secondary fatty acids (compound 406 or LA-14-PP)
is virtually inactive in in vitro assays using human cells
(1) . LPS molecules bearing tetraacyl lipid A structures may be
generated by a leukocyte enzyme, acyloxyacyl hydrolase
(2) ,
which removes secondary fatty acyl chains from a variety of bacterial
LPSs
(3) . Enzymatically deacylated LPS (dLPS) has greatly
reduced stimulatory activity in human cells. Moreover, Pohlman et
al. (4) showed that dLPS inhibits the ability of LPS, but
not other agonists, to stimulate adhesion of neutrophils to human
endothelial cells. Subsequently, others showed that dLPS
(5, 6, 7, 8) and tetraacyl lipid A
partial structures (derived either by purification of biosynthetic
precursors
(7, 9, 10) or by chemical synthesis)
(11, 12) can inhibit a variety of LPS-induced responses
in human monocyte/macrophages, neutrophils, endothelial cells, and
whole blood.
H]LPS and [
H]dLPS to
THP-1 cells under conditions that prevent ligand internalization. Our
results indicate that, depending upon the experimental conditions, dLPS
can prevent the LPS-CD14 interaction by competing with LPS for binding
either LBP or CD14. In addition, dLPS can also antagonize the
stimulatory activity of LPS without inhibiting the binding of LPS to
CD14. Remarkably, although dLPS binds to CD14 with substantially lower
affinity than does LPS, 50% inhibition of the response to LPS can occur
with dLPS:LPS ratios of 0.2 or less. Our results indicate that LPS
antagonism by lipid A analogs may occur at three distinct sites in the
LPS recognition pathway, and they point to the existence of an
undiscovered inhibitory mechanism that occurs after the interaction of
LPS with CD14.
Cells
Cells of the THP-1 human monocyte cell
line
(21) were obtained from D. Altieri (Scripps Research
Institute, La Jolla, CA) and cultured as described previously
(8) . To induce elevated expression of CD14, the cells were
exposed to 0.05 µM 1,25-dihydroxyvitamin Dfor
48 h.
Reagents
Purified LBP from acute phase rabbit
serum was generously provided by Dr. Peter S. Tobias (Scripps Research
Institute, La Jolla, CA), and each lot was tested to determine the
ratio of LBP to LPS that produced maximal binding to CD14. Unless
otherwise stated, a molar and functional excess of LBP was used in the
binding assays. On a weight basis, LBP was typically present at
15-80-fold higher concentrations than LPS. Proteinase K,
phosphatidylinositol-specific phospholipase C (PI-PLC from Bacillus
cereus), Nonidet P-40, sodium deoxycholate, SDS, iodoacetamide,
phenylmethylsulfonyl fluoride, aprotinin, leupeptin, TPCK, and ZPCK
were obtained from Sigma. Octylglucoside
(octyl--D-glucopyranoside) was from Calbiochem (San
Diego, CA). Anti-CD14 mAbs 60bca and 26ic in mouse ascites fluid were
provided by Dr. Robert F. Todd (University of Michigan, Ann Arbor).
LPS Preparations
[H]LPS was
biosynthetically labeled using Escherichia coli LCD25
(22) to a specific activity of 6
10
dpm/µg of LPS. A molecular weight of 4,000 was estimated for
the LPS monomer
(8) . Unlabeled LPS was derived from the same
strain, LCD25. dLPS was produced from these preparations using
acyloxyacyl hydrolase as described previously
(5) . The specific
activity of [
H]dLPS was 4.2
10
dpm/µg. LPS from Salmonella minnesota R5 was
repurified using deoxycholate
(23) , derivatized with SASD and
radioiodinated using Iodogen (Pierce) as described elsewhere
(24) . This preparation did not contain protein impurities
(<0.1% by weight) by silver staining or by fluorography of SDS-PAGE
gels.
Preparation of Fab Fragments of mAb 60bca
60bca
was purified on -bind G (Pierce) before preparation of antibody
fragments. Fab fragments were purified from papain digests using
protein A-Sepharose (unbound fraction) followed by gel filtration
chromatography on Sephacryl S-100-HR (Sigma). Preparations were pure as
assessed by Coomassie Blue-stained SDS-PAGE gels.
Measurement of CD14 Expression
CD14 expression was
measured by analyzing the binding of mAb 60bca (Fab) as described
previously
(25, 26) . Briefly, cells were incubated with
increasing concentrations of radioiodinated antibody fragments for 1 h
on ice. Cell-bound and free antibody fragments were separated by
centrifugation over a 10% sucrose cushion. Nonspecific binding was
determined in the presence of unlabeled 60bca (whole IgG) at a 150-fold
excess over the highest concentration of labeled antibody used. The
data were analyzed by hyperbolic curve fitting or Scatchard analysis
(below). The number of molecules/cell was derived from specific
radioactivities determined by measuring labeled Fab concentrations
spectrophotometrically and assuming a molecular weight of 50,000.
LPS Binding Assay
Equilibrium binding of
[H]LPS to the cells was performed by the method
of Kirkland et al. (26) with minor modifications.
THP-1 cells were washed in ice-cold HNE buffer (20 mM HEPES,
pH 7.4, 150 mM NaCl, 1 mM EDTA), preincubated for 30
min at 37 °C in SEBDAF buffer (20 mM HEPES (pH =
7.4), 150 mM NaCl, 1 mM EDTA, 300 µg/ml BSA, 10
mM NaN
, 2 mM NaF, 5 mM
deoxyglucose) to prevent ligand internalization. The cells were then
centrifuged, resuspended in either SEBDAF buffer or RPMI-B (RPMI 1640
containing 300 µg/ml BSA, 10 mM NaN
, 2
mM NaF, and 10 mM deoxyglucose), and counted using a
hemocytometer. Cells used for determination of nonspecific binding were
preincubated for at least 15 min on ice with mAb 60bca (usually 0.7
µl of ascites fluid in 50-100 µl of binding buffer)
before adding LPS. The LPS was sonicated
(8) either in SEBDAF
buffer or RPMI-B, and purified LBP was added to the LPS at room
temperature 10 min before mixing with the cells. In some experiments
the LPS and LBP were added to the cells at the same time, with similar
results. The LPS (in 10-20 µl) was added to the cells (4
10
VD3-induced cells in 50 µl, final volume)
and allowed to bind with frequent mixing for 30 min at 37 or 10 °C.
450 µl of ice-cold binding buffer (without inhibitors) were then
added, and the mixtures were centrifuged at 6,000 rpm (2,800
g) for 15 s. The
H contained in 200 µl of
supernatant was counted to determine the amount of unbound LPS, the
remaining supernatant was aspirated, and the cells were washed with 500
µl of binding buffer (without inhibitors). The cells were
resuspended in 100 µl of buffer, and the cell-associated
H was counted after adding 0.2 ml of 1% SDS, 10 mM
EDTA and 3 ml of scintillation fluid. The time required to wash the
cells resulted in minimal dissociation since the t for ligand
dissociation was greater than 1 h (not shown). The previously described
(26) wash step (30 min in 50 µl of binding buffer) was
omitted in most experiments since this step had little or no effect on
the results of the assay. Fewer than 10% of the cells were permeable to
trypan blue following incubation in SEBDAF or RPMI-B.
Data Analysis
Kand
B
values were determined by curve fitting to the
hyperbolic function ( B
[free
LPS]/( K
+ [free LPS])
using GraphPad InPlot Software (Graph Pad Software Inc., San Diego, CA)
or by Scatchard analysis. The K
, or
equilibrium dissociation constant, is defined as the concentration of
free ligand required to saturate half of the available CD14 binding
sites. B
refers to the maximum number of ligand
binding sites under saturating conditions.
Stripping Cell Surface-bound Ligands
Labeled LPS
or dLPS were bound to the cells and washed as described above. The
surface-bound ligands were then stripped by two methods. (i) The cells
were incubated on ice for 45 min in 500 µl of 0.02% proteinase K in
phosphate-buffered saline
(27) , 500 µl of buffer containing
20% fetal calf serum were added, the cells were recovered by
centrifugation, and the radioactivity in an aliquot of supernatant was
counted. (ii) Stripping with PI-PLC was performed as previously
described
(26) using 0.5 unit of PI-PLC with the washed cells
in a 50-µl final volume for 30 min at 10 °C. The proportions of
tritiated ligands in the cells and culture supernatants were determined
by scintillation counting. In some experiments, acid stripping of the
ligands was attempted by resuspending the washed cells in ice-cold
phosphate-buffered saline adjusted to pH 3 with acetic acid for 5 min,
followed by centrifugation to recover the cells and supernatants.
Cross-linking LPS to CD14
VD3-induced THP-1 cells
(5 10
cells in 10 ml of RPMI 1640 + 1% fetal
calf serum) were incubated in the dark for 10 min at 37 °C with 20
ng/ml
I-ASD-LPS in the presence or absence of the same
concentrations of unlabeled dLPS added 1 min before the LPS. The cell
suspension was transferred to a 10-cm dish on ice, and the cross-linker
was activated for 2 min with a hand-held 254-nm UV light (UVP, San
Gabriel, CA). The cells were washed in cold phosphate-buffered saline
and lysed for 30 min on ice in 200 µl of lysis buffer (20
mM Tris(Cl
), pH 7.5, 250 mM NaCl, 2
mM EDTA, 20 mM octylglucoside, 1% (w/v) Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 5 mM iodoacetamide, 1
mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1
µg/ml leupeptin, 0.1 mM TPCK, and 0.1 mM ZPCK).
The lysate was centrifuged for 15 min at 12,000
g, and
200 µl (4 mg protein) of lysate supernatant were passed over a
50-µl column of protein A-Sepharose containing prebound
CD14
mAb 26ic. The column was washed with 1 ml of lysis buffer followed by
200 µl of lysis buffer diluted 1:10 in 100 mM Tris, pH
6.8, 2 mM EDTA, and 10% glycerol. The CD14 was recovered by
adding 50 µl of 2
SDS sample buffer to the protein
A-Sepharose, heating in boiling water for 5 min, and removing the beads
by centrifugation. The samples were run on a 16-cm, 10% polyacrylamide
SDS-PAGE gel, and the CD14 bands were detected by autoradiography.
Cell Stimulation and Interleukin-8 (IL-8)
Assays
VD3-induced THP-1 cells were washed in cold HNE buffer
and resuspended in RPMI 1640 + 300 µg/ml BSA at 0.8
10
cells/100 µl, final volume, in microcentrifuge
tubes. For comparison with cross-linking assays, the cells were
resuspended in RPMI 1640 + 1% fetal calf serum at 5
10
cells/ml in six-well culture plates. LPS or dLPS were
prepared in RPMI + 300 µg/ml BSA by sonication (see above),
and LBP or 1% fetal calf serum was added at room temperature 10 min
before adding to the cells. The cells were incubated for the specific
times at 37 °C, pelleted by brief centrifugation, and IL-8 was
measured in the culture supernatants by enzyme-linked immunosorbent
assay (R & D Systems, Minneapolis, MN).
THP-1 Cells Express CD14
THP-1 cells cultured in
the presence of VD3 express elevated levels of cell-surface CD14
(8, 28) as determined by fluorescence-activated cell
sorting analysis. Quantitative analysis of CD14 expression was
performed by binding I-labeled Fab fragments of an
CD14 mAb to cells that had been exposed to VD3 for 48 h. This
analysis revealed an average of 5.2 (± 2.3 S.D., n = 5)
10
binding sites/cell
( K
= 10
M), in good agreement with previous estimates (3
10
and 6
10
sites/cell)
(25, 26) made under similar conditions using a different
CD14
mAb. Uninduced cells expressed low levels of CD14 that were
undetectable by fluorescence-activated cell sorting (1.3 (± 0.6
S.D.)
10
binding sites/cell, n =
7)), also in agreement with a previous report
(25) .
Pretreatment of Cells in SEBDAF Buffer Inhibits
Internalization of LPS and dLPS at 37 °C
To study binding of
LPS or dLPS to the cell surface in the absence of ligand
internalization, THP-1 cells were preincubated with azide,
deoxyglucose, and fluoride (SEBDAF buffer) to deplete cellular ATP.
Preincubation for 30 min at 37 °C in SEBDAF buffer did not result
in significant cell loss or permeability to trypan blue. Subsequent
binding incubations were done in SEBDAF buffer or in RPMI 1640 that
contained the same inhibitors (RPMI-B). Ligand binding to
SEBDAF-pretreated cells was performed at 10 or 37 °C. Two methods
were used to strip the surface-bound ligands from the cells. (i) PI-PLC
cleaves CD14 from its lipid anchor
(26) and (ii) proteinase K
digests unprotected proteins on the extracellular surface of the plasma
membrane
(27) . As shown in , the percentages of
cell-bound ligands released by either method were only modestly
decreased following binding at 37 °C compared to binding at 10
°C. This indicates that internalization was minimal at 37 °C.
Treatment of the cells with PI-PLC removed most of the bound LPS and
dLPS, providing evidence that the ligands remained bound to CD14. When
the metabolic inhibitors were not added, most of the bound LPS or dLPS
became internalized ( i.e. could not be stripped)
(). In separate experiments (not shown), proteinase K
treatment did not significantly reduce the cell number or increase
cellular permeability to trypan blue. However, this treatment destroyed
many of the cells that were already trypan blue-permeable (10% or less)
before proteinase K treatment. In experiments not shown, LPS and dLPS
were not removed by a conventional stripping protocol for protein
ligands (acetate-buffered saline at pH 3) even though this treatment
dramatically increased cell permeability to trypan blue. The rate of
spontaneous dissociation during the stripping procedures was consistent
with the slow, monophasic dissociation rates for LPS and dLPS
( tgreater than 1 h) (data not shown),
suggesting that these ligands dissociate from CD14. Taken together, the
data in indicate that, under the conditions of the binding
assay, LPS and dLPS bind to CD14 with minimal internalization and that
most of each ligand remains associated with CD14 after 30 min at 37 or
10 °C.
Specific Binding of LPS and dLPS to THP-1 Cells Is
CD14-dependent
An incubation temperature of 37 °C was used
for most experiments since internalization was not significantly
increased at this temperature compared to 10 °C. As shown in
Fig. 1
, most of the cellular binding of
[H]LPS or [
H]dLPS was
inhibited by a large excess of unlabeled LPS or dLPS. The absence of
LBP or the presence of anti-CD14 mAb 60bca also inhibited specific
binding, indicating that LBP and CD14 mediate essentially all
measurable specific binding in these cells. As shown previously
(8) , a small but measurable amount of LPS binding to CD14
occurred in the absence of LBP. Similar results were obtained when
ligands were bound to the cells in the presence of SEBDAF buffer (not
shown). Analysis of [
H]LPS and
[
H]dLPS Binding to CD14-The specific
binding of LPS and dLPS to CD14 was saturable when studied in SEBDAF
buffer (Fig. 2, A and B). In the presence of
excess LBP, the apparent equilibrium dissociation constant
( K
) for LPS was 33 ng/ml (± 1,
n = 2) or 8.3 nM. This value is similar to
those reported by Kirkland et al., who used human serum as a
source of LBP at 10° or 37 °C ( K
= 27-49 nM). Analysis of dLPS binding
(Fig. 2 B) showed a lower binding affinity, as reflected
in a higher K
(101 ng/ml (± 14,
n = 2) or 28 nM). The results obtained by
Scatchard analysis were similar (not shown). dLPS binding in RPMI-B
(not shown) yielded results that were similar to those obtained in
SEBDAF buffer. In RPMI-B, LPS binding was more erratic, presumably due
to increased LPS aggregate structure in the presence of divalent
cations, and could not be analyzed with confidence. The analysis of LPS
and dLPS binding affinities in uninduced THP-1 cells (not shown)
yielded similar results to those obtained in VD3-induced cells.
Figure 1:
Binding of [H]LPS
and [
H]dLPS to THP-1 cells. VD3-induced THP-1
cells were preincubated in SEBDAF buffer, and
[
H]LPS or [
H]dLPS (25
ng/ml) was bound to the cells (4
10
cells in 50
µl RPMI-B) with a molar excess of purified LBP (375 ng/ml) at 37
°C as described under ``Materials and Methods.'' Specific
binding was inhibited by mixing a 100-fold excess of unlabeled
competitor (100
LPS or 100
dLPS) with the labeled
ligand, preincubating for 10 min at room temperature with LBP, and
adding this mixture to the cells. Binding was also measured in the
absence of LBP ( No LBP), and binding to CD14 was inhibited by
preincubating the cells with mAb 60bca for 15 min before adding the
labeled ligands ( CD14 mAb). Standard deviations (not shown)
were within 10% of the means of triplicate
determinations.
Figure 2:
Analysis of LPS and dLPS binding.
VD3-induced THP-1 cells were pretreated in SEBDAF buffer, and the
binding of increasing concentrations of [H]LPS
( A) or [
H]dLPS ( B) was measured
in the presence of excess LBP. Assays were performed in triplicate with
4
10
cells/50 µl of SEBDAF buffer at 37 °C.
Specific binding is shown by filled circles and error bars which denote the mean ± S.D. of triplicate determinations.
Nonspecific binding ( dashed lines) was determined in the
presence of
CD14 mAb 60bca.
Bvalues (maximal binding at saturation) for
LPS and dLPS (Fig. 2, A and B) were somewhat
variable but always within the same order of magnitude as
B
values obtained from the binding of
I-Fab fragments of CD14 mAb 60bca (above). This suggests
that the binding stoichiometry of LPS (or dLPS) to CD14 is near 1:1, in
contrast to previously reported values of 8:1 and 20:1
(26) .
When LBP Is Limiting, Low Concentrations of dLPS Can
Inhibit LPS Binding to CD14
When the LBP concentration is
suboptimal, concentrations of dLPS that are far below its
Kcan inhibit binding of LPS to CD14. As
shown in , the binding of 2 nM
[
H]LPS (8 ng/ml) was submaximal in the presence
of 0.1 nM or lower concentrations of LBP. Under these
conditions, adding an equal concentration of dLPS inhibited LPS binding
to CD14 by approximately 50%, whereas this amount of dLPS had very
little inhibitory effect at LBP concentrations (
1 nM) that
produced maximal LPS binding. The inhibition of LPS binding by dLPS
presumably occurs by competition for binding to LBP.
In the Presence of Excess LBP, High Concentrations of
dLPS Can Inhibit LPS Binding to CD14
As shown in Fig. 3,
50% inhibition of the binding of [H]LPS (3 ng/ml)
occurred in the presence of a concentration of dLPS (approximately 100
ng/ml) that was equal to the binding K
for dLPS. This result was expected since the
K
of a ligand is defined as the
concentration required to occupy half of the available receptor binding
sites at equilibrium. Low concentrations of dLPS (below 10 ng/ml) did
not significantly inhibit LPS binding, since these concentrations are
far below the K
for dLPS. Our data do not
rule out the possibility that dLPS might inhibit the ability of LPS to
bind to CD14 by sequestering the LPS in aggregate structures. This
explanation seems unlikely, however, since sucrose gradient analyses
indicated that both LPS and dLPS were highly disaggregated in the
presence of LBP and SEBDAF buffer (not shown), and the inhibitory
ability of dLPS was similar whether the binding was performed in the
presence (RPMI-B) or absence (SEBDAF) of divalent cations
(Fig. 3).
Figure 3:
Inhibition of [H]LPS
binding to CD14 by high concentrations of dLPS in the presence of
excess LBP. VD3-induced THP-1 cells were pretreated in SEBDAF buffer,
and [
H]LPS (3 ng/ml) was bound to the cells (4
10
cells in 50 µl) in the presence of
increasing concentrations of unlabeled dLPS at 37 °C. Binding was
performed in SEBDAF buffer ( closed circles) or RPMI-B
( open triangles). Error bars denote standard
deviations from the means of triplicate
determinations.
Low Concentrations of dLPS, in the Presence of Excess
LBP, Inhibit LPS-induced IL-8 Production without Inhibiting LPS Binding
to CD14
To compare binding with function, we studied the effects
of LPS and dLPS on the production of IL-8 under similar conditions used
for the binding assay without the metabolic inhibitors. As shown in
Fig. 4
, the IL-8 response was maximal (EC) at
1-3 ng/ml LPS and half-maximal (EC
) at approximately
0.2 ng/ml. (The assay measures the release of IL-8 into the culture
supernatant after a 2-h incubation. In experiments not shown, the cells
continued to release IL-8 at a slower rate after 2 h but also with the
maximal response at a dose of approximately 1 ng/ml.) The EC
(0.2 ng/ml or 5
10
M) and
EC
(1-3 ng/ml) are far below the
K
for LPS binding (33 ng/ml or 8
10
M), indicating a large spare receptor
effect
(29) with respect to CD14 binding.
Figure 4:
LPS dose response for IL-8 induction.
VD3-induced THP-1 cells (8 10
in 100 µl, final
volume, of RPMI 1640 + 300 µg/ml BSA) were incubated at 37
°C with increasing concentrations of LPS in the presence of excess
LBP for 2 h at 37 °C. IL-8 in the culture medium was measured as
described under ``Materials and Methods.'' Closed circles and bars denote means and ranges of duplicate
determinations.
dLPS did not
stimulate IL-8 release and strongly inhibited this response to LPS. As
shown in Fig. 5, a brief preincubation with dLPS almost
completely suppressed the response to an equal amount of LPS.
Substoichiometric concentrations of dLPS (with respect to LPS) were
also very inhibitory even when added simultaneously with LPS (not
shown), in keeping with the previously observed effects of dLPS on
LPS-induced NF-B, IL-1
(8) , and protein tyrosine
phosphorylation (data not shown). The effects of varying concentrations
of dLPS on the LPS dose response (Fig. 5) are difficult to
analyze since they show characteristics of noncompetitive inhibition
( i.e. maximal control levels of activity are not attained with
increasing LPS concentrations in the presence of dLPS) and competitive
inhibition (a lower but approximately equal plateau of activity occurs
with increasing LPS concentrations in the presence of each
concentration of dLPS). Most importantly, dLPS inhibits maximal
stimulatory concentrations of LPS at dLPS concentrations that are far
below its binding K
without significantly
inhibiting LPS binding to CD14.
Figure 5:
Inhibition of LPS-induced IL-8 by dLPS.
Incubations were performed as described in Fig. 4 except that dLPS
(with excess LBP) was added 10 min before the addition of LPS. No dLPS,
closed circles; 0.1 ng/ml dLPS, open circles; 0.4
ng/ml dLPS, open triangles; and 1 ng/ml dLPS, open
squares. Data points and error bars denote means and
ranges of duplicate determinations.
dLPS Inhibits LPS Function without Inhibiting
Cross-linking of LPS to CD14
To obtain biochemical evidence that
the direct interaction between LPS and CD14 was not inhibited by low
concentrations of dLPS that inhibit LPS responses, we observed
cross-linking to CD14
(25) using LPS that was derivatized with
the photoactivatable cross-linker, SASD. We photoactivated
I-ASD-LPS to CD14 after it had incubated with the cells
for 10 min. Immunoprecipitation of CD14 from detergent lysates showed
that the LPS cross-linked to CD14 (Fig. 6, inset,
lane 1). Cross-linking was not significantly inhibited in the
presence of an equal amount of unlabeled dLPS ( lane 2), but it
was abolished when the cells were preincubated with mAb 60bca ( lane
3). A functional comparison to this experiment was made by
measuring IL-8 release for 90 min under the same conditions. As also
shown in Fig. 6, the addition of dLPS strongly inhibited the IL-8
response under conditions that did not prevent cross-linking LPS to
CD14 on the cells. We conclude that low concentrations of dLPS that do
not block binding of LPS to CD14 can nevertheless antagonize LPS,
presumably at a site that is distal to CD14 binding.
Figure 6:
dLPS inhibits CD14-mediated IL-8 induction
by LPS without inhibiting cross-linking of I-ASD-LPS to
CD14.
I-ASD-LPS was cross-linked to VD3-induced THP-1
cells after binding for 10 min in the presence or absence of the same
amount of unlabeled dLPS. IL-8 release into the culture supernatant was
measured under identical conditions except that the incubations were
continued for 90 min at 37 °C. Data points and error
bars denote means and ranges of duplicate determinations.
Inset, autoradiographs of CD14 immunoprecipitates, analyzed
using SDS-PAGE. Lane 1,
I-ASD-LPS alone;
lane 2, 20 ng/ml unlabeled dLPS +
I-ASD-LPS; lane 3, pretreatment with
CD14
mAb 60bca before binding with
I-ASD-LPS. The arrow indicates the migration position of
CD14.
values. The second
measurement was the EC
for LPS stimulation of IL-8, which
we found to be almost 100-fold lower than the LPS binding
K
, indicating that most of the LPS
molecules that can bind to these cells do not necessarily contribute to
signaling. This phenomenon, called the ``spare receptor
effect,'' has been observed in many known receptor systems
(29) . Large numbers of spare receptors serve to increase the
dose sensitivity of cells to agonists by binding more molecules at low
agonist concentrations. Antagonism that occurs at low concentrations of
agonist and antagonist ( i.e. well below their respective
binding K
values) should involve
mechanisms other than competition for receptor binding.
Competing with LPS for Binding LBP
When the
concentration of LBP is suboptimal for facilitating LPS-CD14 binding,
low concentrations of antagonist can inhibit LPS binding to
cell-surface CD14 (). Competition for binding LBP is the
likely explanation, as Tobias et al. (30) reported
that LPS binds to LBP with a stoichiometry of 1:1, and that the
tetraacyl lipid A structures, lipid IVA and dLPS, inhibit binding of
LBP to immobilized LPS. Competition for limiting amounts of LBP could
be an important inhibitory mechanism in extravascular sites where serum
components are scarce. Competition for LBP may also be an important
factor when in vitro LPS stimulation assays are done with a
limiting concentration of serum in the incubation medium
(11, 12, 31) . Even in the absence of added
serum, the sensitivity of cells to LPS, although mediated largely by
CD14, may depend in part upon residual LBP that remains bound to the
cells after washing
(32) .
Competing with LPS for Binding CD14
When LBP
concentrations are optimal for promoting LPS-CD14 binding, only high
concentrations of antagonist can compete with LPS for binding CD14
(Fig. 3). Approximately 50% inhibition of LPS (3 ng/ml) binding
was observed in the presence of 100 ng/ml dLPS; this is at the dLPS
binding K, or the concentration at which
half of the available binding sites are engaged by dLPS, so inhibition
of LPS binding would be expected at this dLPS concentration. (The
ability of an antagonist to inhibit binding is determined by its
binding K
rather than by the ratio of
antagonist to agonist
(29) .) This mechanism probably accounts
for the ability of high concentrations of lipid A analogs to inhibit
LPS binding to CD14-expressing cells. For example, 300 ng/ml tetraacyl
lipid A analog (PE-4) was required to block the binding of labeled LPS
(30 ng/ml) to human monocytes in the presence of serum
(18) .
Competition for CD14 binding may be the inhibitory mechanism in most
cases in which high analog concentrations are required to block LPS
stimulation.
Competing with LPS at a Site That Is Distal to CD14 in
the Signaling Pathway
As noted above, large numbers of spare
CD14 receptors facilitate maximal cellular responses at ligand
concentrations that are far below the binding Kof either the antagonist or agonist. Competition for binding to
CD14 does not occur at these low concentrations (Fig. 3) because
the ability of the antagonist to inhibit agonist binding competitively
is determined by their respective binding K
values. It has been common practice to determine binding
K
values for receptor antagonists by
measuring the ability of the antagonist to inhibit functional responses
induced by the appropriate agonist
(29) . Our data show that
this method is not appropriate for dLPS, since functional responses to
LPS can be inhibited by dLPS concentrations that are 100-fold below the
K
for dLPS binding. Moreover, these low
concentrations of dLPS can inhibit LPS signaling without blocking LPS
binding to CD14 (Fig. 6). We believe that dLPS is not a CD14
receptor antagonist under these circumstances. Rather, it may inhibit
the interaction of LPS with another molecule that is distal to CD14 in
the signal pathway. Particularly intriguing is the observation that,
despite the lower binding affinity of dLPS for CD14, substoichiometric
concentrations of dLPS can block LPS stimulation.
) of
approximately 2 ng/ml or 500 pM (data not shown). However,
this finding probably does not reflect a saturable subclass of CD14 or
point to the site of dLPS inhibition, since dLPS binds with lower
affinity than LPS and does not significantly inhibit high affinity LPS
binding. Another possibility is that antagonist and agonist molecules
bind to multiple binding sites on the same CD14 molecule (possibly
involving the multiple L XXL XL X motifs on
CD14)
(33) . In this model, the binding of dLPS might produce an
allosteric effect that could inhibit receptor function without
inhibiting the binding of LPS to another site. Data regarding the
existence of multiple LPS binding sites on CD14 are conflicting
(26, 34) , and our data suggest that the binding
stoichiometry between LPS (or dLPS) and CD14 is approximately 1:1.
Moreover, the low number of LPS and dLPS molecules that interact
functionally are not statistically likely to bind to the same molecule
of CD14 since such concentrations are so far below the
K
values for both ligands.
CD14 complex) or after transfer of the
ligands from CD14 to this molecule. Thus, dLPS antagonizes LPS in this
model by interfering with the ability of LPS to interact with the
receptor/effector. This model would also be consistent with the
spare-receptor effect (Fig. 4), as well as with scenarios
proposed to explain how LPS and soluble CD14 (sCD14) stimulate
responses in CD14-negative cells
(35, 36) . The ability
of the proposed molecule to bind the ligands directly (without CD14)
could account for the ability of high concentrations of LPS to
stimulate cells in the presence of antibodies that block CD14 binding
(8, 13, 14, 32) , as well as cells that
express little or no CD14
(27, 37) .
Figure 7:
dLPS
can antagonize LPS at three sites in the LPS recognition pathway:
( 1) by competing for binding LBP, ( 2) by competing
for binding CD14, and ( 3) by interacting at a site distal to
CD14.
Alternatively,
dLPS might antagonize LPS responses by inducing a negative signal that
somehow rapidly inhibits LPS recognition. Our attempts to detect the
signal-inducing ability of dLPS in THP-1 cells have not been revealing.
Some investigators have reported, however, that tetraacyl or other
lipid A partial structures induce priming effects that increase the
sensitivity of cells to LPS
(12, 38, 39) .
Others have reported that low concentrations of a synthetic tetraacyl
lipid A stimulate the migration of human neutrophils
(40, 41) . These data support the notion that tetraacyl
antagonists can deliver signals to cells, offering an interesting
alternative interpretation of their inhibitory mechanism.
Table:
Release
of LPS or dLPS bound to THP-1 cells
H]LPS or
[
H]dLPS (usually 25 ng/ml), in the presence of a
2-fold molar excess of purified LBP in RPMI-B, was allowed to bind at
the indicated temperatures to VD3-induced THP-1 cells that had been
pretreated in SEBDAF buffer. The cells were washed, and the bound
ligands were stripped as described under ``Materials and
Methods.'' The amount of ligand released is expressed as the
percentage of radiolabeled ligand initially bound to the cells. The
data are shown as mean ± S.D. of triplicates (number of
experiments).
Table:
Low concentrations of
dLPS inhibit LPS binding to CD14 when LBP is limiting
10
in 50
µl of RPMI-B) were pretreated in SEBDAF buffer and 2 nM [
H]LPS (8 ng/ml) were then added, alone or
in the presence of 2 nM unlabeled dLPS. The fraction inhibited
by dLPS is expressed as the percent inhibition of LPS bound in the
absence of dLPS. Values are mean ± S.D. of triplicate
determinations.
; TPCK,
N-tosyl-L-phenylalanine chloromethyl ketone; ZPCK,
N-CBZ-L-phenylalanine chloromethyl ketone; BSA,
bovine serum albumin; mAb, monoclonal antibody; PAGE, polyacrylamide
gel electrophoresis; PI-PLC, phosphatidylinositol-specific
phospholipase C; SASD, sulfosuccinimidyl 2-(
p-azidosalicylamido)ethyl-1,3`-dithiopropionate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.