(Received for publication, October 24, 1994; and in revised form, January 4, 1995)
From the
Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.
LPS, ()released from the outer membrane of
Gram-negative bacteria, triggers cells to synthesize and release a
cascade of inflammatory mediators that in vivo may progress to
septic shock(1) . Two effector molecules involved in the LPS
response are LBP and CD14(2) . LBP is a 60-kDa serum
glycoprotein that binds to the lipid A moiety of LPS with a
dissociation constant near 10
M(3) . LBP appears to function as a catalytic
transfer protein in delivering LPS to soluble CD14(4) . LBP
also enhances the binding of LPS to the membrane form of
CD14(5) , although the nature of the LBP interaction with mCD14
has not been evaluated.
Membrane CD14, an LPS receptor, is a 55-kDa glycoprotein attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Because mCD14 does not transverse the membrane, it is not known how the intracellular signal is initiated. Chimera constructs that replaced the GPI anchor with transmembrane domains demonstrate that the anchor is not required for signal transduction(6) . It has been proposed that a yet unidentified transmembrane transducer protein interacts with LPS and mCD14 to transmit the signal(6) . Membrane CD14 is found primarily on myeloid lineage cells and is responsible for the enhanced sensitivity of mCD14-positive cells, including transfected cell lines (7, 8, 9) , to LPS.
In addition to
responding to LPS, cells internalize LPS(10, 11) . The
scavenger receptor has been implicated in the clearance of LPS by
hepatic uptake in mice, RAW 264.7 macrophage cells, and scavenger
receptor-transfected CHO cells(12) . Scavenger receptor
ligands, such as acetylated low density lipoprotein (AcLDL), were shown
to compete in binding and metabolism with lipid IV, the
bioactive precursor of lipid A. However, the ligands were unable to
completely inhibit lipid IV
uptake. LBP and mCD14 also
augment LPS clearance(11) , although any overlap of LBP- and
mCD14-mediated clearance with that of the scavenger receptor has not
been evaluated. Luchi and Munford (11) showed that serum, a
source of LBP, and CD14 enhance but are not absolutely required for LPS
internalization by human neutrophils. It seems likely from these and
other studies that LPS clearance is a separate event from LPS signal
transduction (9, 10, 11, 12) . Here,
we have investigated the surface binding, internalization, and signal
transduction properties of LBP and LPS with the model system of
CD14-transfected CHO cells to further define the mechanism of
LPS-initiated cellular activation.
Figure 1:
Binding profile of LBP to CHO cells. S-Labeled LBP (40 nM) incubated at 37 °C with
or without Re595 LPS (40 nM) was added to cells, and complexes
were allowed to form for 40 min at 4 °C. The anti-CD14 antibody MY4
(10 µg/ml) was added to CHO-hCD14 cells for 10 min at 4 °C
prior to addition of LBP and LPS. LBP binds to CHO cells only when both
mCD14 and LPS are present.
LBP binding to mCD14 is dependent not only on LPS but also on the
relative amount of LPS to LBP. As shown in Fig. 2, the optimal
LPS concentration for maximum association of S-labeled LBP
with cells occurs at approximately 1:1 to 1:2 LBP-LPS stoichiometry.
Very high levels of LPS block
S-labeled LBP binding to
cells. If LPS simply was binding to mCD14 in competition with
LBP
LPS complexes, then one would expect the amount of mCD14
present to effect the optimal LPS concentration for LBP binding.
However, we observed that even though the amount of
S-labeled LBP bound increased with increasing mCD14, the
LPS concentration that yields maximum
S-labeled LBP
binding remained the same (see Fig. 2). We evaluated up to a
10-fold range in cell number without observing a shift in the
S-labeled LBP binding maximum (data not shown).
H-Labeled LPS binding to cells also reaches a maximum at
approximately 1:1 to 1:2 LBP-LPS stoichiometry(15) . However,
H-labeled LPS binding does not return to background levels
when excess LBP is present but rather reaches a plateau(15) .
We evaluated a 100-fold molar excess of LBP to
H-labeled
LPS without observing a reduction in
H-labeled LPS binding
(data not shown).
Figure 2:
Effects of LPS on LBP binding to
CHO-hCD14. S-Labeled LBP incubated at 37 °C with
increasing amounts of Re595 LPS subsequently was incubated with cells
for 40 min at 4 °C. An LBP binding maximum is observed when the LBP
to LPS molar ratio is approximately 1:1 to 1:2. The binding maximum is
dependent on LBP concentration but not on cell
number.
Figure 3:
Binding saturation. Panel A evaluates LBP binding to CHO-hCD14 cells as a function of LBP when
the LBP to LPS molar ratio is held constant at 1:1. Equal molar amounts
of S-labeled LBP and Re595 LPS were incubated with 1.5
10
CHO-hCD14 cells for 40 min at 37 °C in the
presence of inhibitors to internalization. No binding saturation is
observed. Panel B shows binding saturation when the LBP
concentration was held constant at 12 nM; panel C shows saturation of both
S-labeled LBP and
H-labeled LPS binding when the LPS concentration was held
constant at 12 nM. Complexes in panel A and panel
B were formed by incubating
S-labeled LBP and
H-labeled LPS with 5
10
CHO-hCD14 cells
for 20 min at 37 °C with inhibitors to
internalization.
Figure 5:
PI-PLC cleavage of mCD14 GPI tail releases
bound LBP. 1 10
cells were incubated with 220
nM
S-labeled LBP + 220 nM Re595 LPS
for 40 min at either 4 or 37 °C. Cells then were incubated with or
without PI-PLC for 20 min at 12 °C. Cells were pelleted, and the
supernatant was assayed for CD14 by ELISA. The pellet was counted to
determine cell-associated LBP.
Figure 4:
LBP and LPS bind mCD14 with similar
kinetics. 10 nMS-labeled LBP and 50 nM
H-labeled LPS were incubated with 5
10
cells at 37 °C in binding buffer with inhibitors to
internalization. Panel A is a plot of the natural logarithm of
the amount of ligand bound at equilibrium (F(
)) minus
ligand bound at time t (F(t)). From the slopes of the
curves, LBP and LPS have the same association half-life of 1.2 ±
0.1 min. Panels B and C show that even though the LBP
to LPS binding stoichiometry is approximately 1:4, LPS and LBP reach
equilibrium together.
We next evaluated the
resistance of S-labeled LBP and
H-labeled LPS
binding to dissociation by protease as a function of time at 37 °C. Fig. 6shows that the resistance of both
S-labeled
LBP and
H-labeled LPS binding to release increases at
1%/min. This is similar to a value determined for the rate of LPS
internalization by human neutrophils(11) . Internalized
S-labeled LBP also should be stable to conditions favoring
its dissociation. As shown in Fig. 7, 35% of the
S-labeled LBP is resistant to dissociation after dilution
into a 300-fold excess of unlabeled LBP. However, when inhibitors to
internalization are added, only 10% is resistant to dissociation. The
inability to completely dissociate
S-labeled LBP by
PI-PLC, protease, or unlabeled LBP suggests that LBP is internalized
with LPS. Since the amount of mCD14 released by PI-PLC is not affected
by incubation conditions, mCD14 either recycles to the cell surface or
is not directly involved in LBP
LPS complex internalization.
Figure 6:
LBP and LPS binding to CHO-hCD14 cells
becomes protease resistant after incubation at 37 °C. S-Labeled LBP (25 nM) and
H-labeled
LPS (25 nM) were incubated with 5
10
CHO-hCD14 cells for 20 min at 4 °C. Samples were shifted to
37 °C for the indicated time and returned to 4 °C. Proteinase K
(0.4 mg/ml) was added at 4 °C for 20 min. Cells were pelleted,
washed once in binding buffer, and counted to determine
protease-resistant LBP and LPS binding.
Figure 7:
Effect of 18E12 and AcLDL on S-labeled LBP resistance to dissociation. 5
10
cells were incubated with
S-labeled LBP (20
nM) and
H-labeled LPS (10 nM) for 30 min
at 37 °C. 18E12 (10 µg/ml), AcLDL (100 µg/ml), or
inhibitors to internalization were added to cells before addition of
radiolabeled LBP and LPS. Unlabeled LBP then was added as described
under ``Experimental Procedures.''
S-Labeled LBP
resistant to displacement by LBP is
plotted.
18E12 inhibits LPS-stimulated TNF release as well as other cellular
responses with little effect on LPS binding(15) . Fig. 7shows that 18E12 does not inhibit S-labeled
LBP internalization. Hampton et al.(12) demonstrated
that LPS interacts with scavenger receptor-transfected CHO cells and
that this interaction is blocked by AcLDL. To test the possibility that
scavenger receptor endogenous to CHO cells is involved in LBP
LPS
complex internalization, AcLDL was added to the cells prior to LPS and
LBP binding. AcLDL had little effect on either the extent of
LBP
LPS complex binding (data not shown) or on LBP resistance to
dissociation (see Fig. 7). Thus, inhibition of LPS-dependent
signaling by 18E12 does not inhibit LBP internalization nor does AcLDL,
an LPS-competitive ligand for the scavenger receptor.
Figure 8:
Effect of antibodies on binding and cell
activation. The bargraph on the left illustrates the effect of the indicated antibodies (10 µg/ml)
on binding of S-labeled LBP (36 nM) and
H-labeled LPS (72 nM) to 5
10
CHO-hCD14 cells (4 nM). Anti-CD14 antibodies were added
to cells for 10 min at 4 °C prior to addition of LBP
LPS
complexes; anti-LBP antibodies were added to LBP
LPS complexes for
10 min at 4 °C prior to addition of cells. Complexes were formed
for 30 min at 37 °C in binding buffer. 18G4 reduced LBP binding to
background levels, whereas LPS binding was observed at 10 ± 2%
above background; 18E12 reduced LBP and LPS binding only slightly to 90
± 3% of the controls. 2B5 and 28C5 reduced binding to background
levels. The bargraph on the right illustrates the effect of the indicated antibodies (10 µg/ml)
on NF-
B activation of CHO-hCD14 cells by Re595 LPS (100 ng/ml).
The control antibody and 18G4 did not diminish NF-
B activation,
whereas 2B5, 18E12, and 28C5 were
inhibitory.
The binding of S-labeled LBP to cells, only in
the presence of mCD14 and LPS, provides direct evidence for the
formation of an LBP
LPS
mCD14 ternary complex. This is
congruent with the earlier work of Wright et al.(23) ,
which showed that LBP binding to macrophages is LPS dependent. The
optimal LBP to LPS stoichiometry for LBP binding to mCD14 is
approximately 1:1 to 1:2, which agrees with the stoichiometry observed
for LBP binding to LPS immobilized on plastic microtiter
plates(3) . Stoichiometry estimates are approximate because LPS
is heterogeneous in size(24) . However, even when using the
same LPS preparation, variable amounts of LPS associate with LBP and
mCD14 when the LBP to LPS molar ratio is varied. This implies that in
addition to LPS monomers, LPS aggregates bind to LBP and mCD14. An
alternative explanation for variability in stoichiometry is that LBP
has multiple LPS binding sites. However, inspection of its primary
sequence does not reveal any repeated motifs. Further characterization
of the LBP structure will aid in distinguishing between these
possibilities.
LPS is highly aggregated in aqueous solutions with
monomer solubility in the range of 10M(26) . Incubation of LPS with LBP alters its
aggregation properties. Size analysis of LBP and LPS by sucrose
gradients shows that 1) incubation of LBP with excess LPS increases the
apparent size of LBP to co-sediment with the aggregated LPS and 2)
incubation of stoichiometric amounts of LBP and LPS reduces the
apparent size of LPS to co-sediment with LBP near the apparent size of
LBP alone. (
)When binding to cells is evaluated, LPS
concentrations in large excess to LBP inhibit LBP binding to mCD14.
While it is possible that LPS directly competes with LBP
LPS
complexes for mCD14, insensitivity of optimal LBP binding to cell
number and hence mCD14 concentration suggests that LPS aggregation of
LBP is the primary factor. One phenomenon that is difficult to explain
from LBP and LPS size analysis in solution is the inability to saturate
LBP
LPS complex binding to mCD14 (i.e.Fig. 5A). Whereas equal molar amounts of LBP and
LPS are not observed to aggregate in solution, they do accumulate via
mCD14 on the cell surface. The flexibility in the LBP
LPS complex
stoichiometry may enable LBP to accommodate in vivo fluctuations in LPS concentration, while the ability of mCD14 to
sequester large numbers of LBP
LPS complexes may provide for
efficient clearance of LPS, especially where local LPS concentrations
are high.
At first glance, the mechanism by which LBP interacts with
mCD14 appears distinct from its interaction with sCD14. Whereas LBP
catalyzes the transfer of LPS to sCD14(4) , LBP associates with
mCD14 as an LBPLPS complex. This leads us to conclude that a
membrane component, distinct from mCD14, forms a weak interaction with
LBP to stabilize its association with the cell. Stabilization cannot be
attributed to LBP interactions with the GPI tail of mCD14 since in
studies not shown LBP binding is observed in 70Z/3-hCD14CI cells (6) that express a CD14 transmembrane construct. It is likely
that 18G4 binds to the LBP epitope involved in the stabilization. Since
18G4 has no effect on the transfer of LPS to sCD14 or cell activation
through mCD14, LBP-facilitated binding of LPS to mCD14 must occur even
in the presence of 18G4. Whereas LBP binding is at background levels in
the presence of 18G4, 10% of LPS binds, and cell activation ensues.
This behavior suggests that transfer of LPS to mCD14 by LBP in the
presence of 18G4, which resembles LBP and LPS interactions with sCD14,
is sufficient for cell activation.
Internalization of LPS has been
characterized in numerous systems, and it now seems evident that both
LBP and mCD14 participate in this process. Scavenger receptors have
been identified as binding LPS(12) , but our results suggest
that internalization initiated by first anchoring to mCD14 occurs
independently of the scavenger receptor. In our model, LBP delivers LPS
to the cell surface through formation of a ternary
LBPLPS
mCD14 complex. Internalization and signal
transduction pathways diverge at this point. Fig. 9illustrates
how the antibodies 18E12 and 18G4 separate the pathways. In signal
transduction, LPS and mCD14 interact with a putative transducer to
initiate an intracellular response. 18E12 is thought to block the
LPS-mCD14 transducer interaction and thus prevent cellular activation
by LPS. There is no evidence that LBP is directly involved in the
transducer activation. Indeed, the ability to eliminate LBP binding
without impeding signaling implies that it is not. In the
internalization pathway, LBP stays closely associated with LPS, whereas
mCD14 either remains or returns to the cell surface. 18G4 is thought to
inhibit LBP and LPS internalization by preventing LBP and LBP
LPS
complexes from accumulating on the cell surface.
Figure 9:
Internalization and signal transduction
utilize LBP and mCD14. LBP binds LPS in serum and delivers it to mCD14
on the cell surface. Some complexes initiate cellular activation,
perhaps through a transmembrane transducer molecule, whereas other
complexes are internalized. Panel A illustrates the effects of
18E12. 18E12 allows binding and internalization of LBPLPS
complexes but blocks the epitope on mCD14 involved in signal
transduction, presumably the LPS-mCD14 transducer interaction. Panel B illustrates the effects of 18G4. LBP association with
mCD14 is stabilized through a weak interaction with an unidentified
membrane component. 18G4 recognizes the LBP epitope involved in this
interaction and prevents LBP binding and internalization. 18G4 does not
affect transfer of LPS to mCD14 and subsequent cellular
activation.
Maximal LPS binding
occurs when LBP is not limiting. During an acute phase response, LBP
serum levels rise from 0.1-5 µg/ml to 50
µg/ml(23, 25) . It is possible that while there is
sufficient LBP present in serum to initiate signaling by LPS and
thereby initiate an acute phase response, LBP levels rise to ensure
efficient clearance of LPS from circulation. LBP is synthesized by
hepatocytes(23, 25) , and LPS is cleared by the
liver(12) . Future research may show that hepatic macrophages
(Kupfer cells) facilitate clearance of LPS in an LBP- and
mCD14-dependent manner. Indeed, the recent identification of mCD14
up-regulation in response to LPS in mouse Kupfer cells (27) supports the hypothesis that LBP and mCD14 are involved in
LPS clearance. Any coordinated effect of the scavenger receptor with
LBP and mCD14 awaits further investigation. In conclusion, the LPS
signal transduction and internalization pathways are optimal when both
LBP and mCD14 participate and diverge after the initial
LBP
LPS
mCD14 ternary complex forms.