(Received for publication, February 21, 1996)
From the
Phospholipid transfer protein (PLTP) and lipopolysaccharide-binding protein (LBP) are lipid transfer proteins found in human plasma. PLTP shares 24% sequence similarity with LBP. PLTP mediates the transfer and exchange of phospholipids between lipoprotein particles, whereas LBP transfers bacterial lipopolysaccharide (LPS) either to lipoprotein particles or to CD14, a soluble and cell-surface receptor for LPS. We asked whether PLTP could interact with LPS and mediate the transfer of LPS to lipoproteins or to CD14. PLTP was able to bind and neutralize LPS: incubation of LPS with purified recombinant PLTP (rPLTP) resulted in the inhibition of the ability of LPS to stimulate adhesive responses of neutrophils, and addition of rPLTP to blood inhibited cytokine production in response to LPS. Transfer of LPS by rPLTP was examined using fluorescence dequenching experiments and native gel electrophoresis. The results suggested that rPLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to reconstituted HDL particles, but it did not transfer LPS to CD14. Consonant with these findings, rPLTP did not mediate CD14-dependent adhesive responses of neutrophils to LPS. These results suggest that while PLTP and LBP both bind and transfer LPS, PLTP is unable to transfer LPS to CD14 and thus does not mediate responses of cells to LPS.
Lipopolysaccharide (LPS(); endotoxin) is a membrane
lipid of Gram-negative bacteria that acts as a potent inflammatory
stimulus in humans and other mammals(1) . Recent work has
suggested that a plasma protein called LPS-binding protein (LBP) is
important in trafficking LPS in blood. LBP can transfer LPS to
lipoprotein particles, resulting in the functional neutralization of
LPS(2) . LBP also mediates functional responses of cells to LPS (3) by facilitating the transfer of LPS to CD14(4) , a
glycoprotein found both as a soluble monomer in the blood (soluble
CD14, sCD14) and as a glycosylphosphatidylinositol-linked membrane
protein (mCD14) on monocytes, macrophages, and neutrophils.
LBP has sequence similarity to two other plasma lipid transfer proteins, phospholipid transfer protein (PLTP, 24% amino acid identity) (5) and cholesteryl ester transfer protein (CETP, 23% amino acid identity)(3) . LBP, PLTP, and CETP are found in plasma associated with HDL particles(2, 6, 7) . CETP facilitates the transfer of cholesteryl esters, triglycerides, and phospholipids between lipoproteins(7) . PLTP mediates the exchange and transfer of phospholipids between lipoprotein particles(6) . PLTP also mediates high density lipoprotein (HDL) conversion, the transformation of HDL into smaller and larger particles(8, 9, 10) . Through these activities, PLTP may regulate HDL level and composition and thereby affect cholesterol metabolism(6) . Because our previous studies indicated that the transfer of LPS may be important in modulating inflammatory responses to LPS, and because LBP and PLTP share sequence similarity as well as related functions, we investigated the ability of PLTP to interact with LPS and transfer LPS to R-HDL or to CD14.
Fluorescence
of BODIPY-LPS was measured as described (16) with an
SLM-SPF500c spectrofluorimeter (SLM Instruments, Urbana, IL).
Time-dependent changes in fluorescence were measured by diluting
BODIPY-LPS and other reagents in PD-EDTA or in PBS (with Ca and Mg
) and quickly mixing in a submicrocuvette
(Starna Cells Inc., Atascadero, CA). Fluorescence emission at 518 nm
was digitally recorded over time, with excitation at 485 nm.
Figure 1:
Neutralization of LPS by rPLTP medium. A, time course of neutralization. LPS (10 ng/ml) was incubated
at 37 °C for the indicated times alone
(-
), with medium from control (nontransfected)
cells (0.5
) (
-
), or with medium from
cells expressing rPLTP (0.5
) (
-
). After
the incubation, neutrophils were then added and adhesion to
fibrinogen-coated plates was measured as described under
``Materials and Methods.'' Adhesion in the absence of LPS was
measured as a control (
-
). B,
neutralization of LPS by rPLTP medium is prevented by anti-rPLTP IgG.
LPS (10 ng/ml) was incubated for 30 min at 37 °C alone
(
-
), with rPLTP medium (0.5
)
(
-
), or with rPLTP medium (0.5
) and the
indicated concentrations of anti-rPLTP IgG
(
-
) or control IgG
(
-
). Neutrophils were then added and adhesion
was measured as in A. Adhesion in the absence of LPS was
measured as a control
(
-
).
To further confirm that PLTP neutralizes LPS and to determine the amount of PLTP needed for neutralization, we measured the effect of purified recombinant PLTP (rPLTP-His) on LPS-induced responses. In a 30-min incubation of rPLTP-His with LPS, complete neutralization of 10 ng/ml LPS was seen with 3 µg/ml rPLTP-His (Fig. 2), and 1 µg/ml rPLTP-His neutralized >90% of the activity of LPS (determined by comparing Fig. 2with a dose-response curve for LPS from the same experiment; not shown). PLTP purified from human plasma also neutralized LPS, with a time course and dose dependence similar to that seen with purified rPLTP-His (not shown).
Figure 2:
Neutralization of LPS by purified
rPLTP-His. LPS (10 ng/ml) was incubated at 37 °C for 30 min alone
(-
) or in the presence of the indicated
concentrations of purified rPLTP-His (
-
).
Neutrophils were then added and adhesion to fibrinogen-coated plates
was measured as described under ``Materials and Methods.''
Adhesion in the absence of LPS was measured as a control
(
-
).
Figure 3: Dequenching of BODIPY-LPS by LBP or rPLTP-His. BODIPY-LPS (40 nM) was incubated at 37 °C in PD-EDTA (A) or PBS (B) alone (curve 1), with human serum albumin (HSA) (40 nM, curve 2), with rPLTP-His (40 nM) (curve 3), or with rLBP (40 nM, curve 4), and the fluorescence emission at 518 nM was measured over time.
The change in fluorescence of BODIPY-LPS caused by rPLTP-His strongly suggests that there is a physical interaction of PLTP with LPS. LBP is known to bind LPS(19, 20, 21) , and LBP and PLTP have similar abilities to cause dequenching of BODIPY-LPS (Fig. 3A). Additionally, several proteins have been identified which neutralize LPS by binding it (see ``Discussion''), and PLTP shares this ability to neutralize LPS ( Fig. 1and Fig. 2). Taken together, these results suggest that PLTP binds LPS, and neutralization of LPS occurs as a result of this binding.
The effect of rPLTP-His or rLBP on the
fluorescence of BODIPY-LPS was less pronounced in the presence of
divalent cations and in the absence of EDTA (Fig. 3B)
than in EDTA-containing buffer (Fig. 3A). In divalent
cation-containing buffer (PBS), rPLTP-His and rLBP consistently caused
a rise in fluorescence of BODIPY-LPS, although only to 1.5 times
the fluorescence of BODIPY-LPS alone (Fig. 3B). This
effect of EDTA is consistent with its ability to destabilize
interactions between LPS molecules (see ``Discussion''). The
experiment shown in Fig. 3A was performed with EDTA to
emphasize the effects of rPLTP-His and rLBP on BODIPY-LPS. Subsequent
experiments, using BODIPY-LPS to assess the transfer activities of PLTP
and LBP, were performed in PBS, taking advantage of the limited effect
of PLTP or LBP alone on the fluorescence of BODIPY-LPS under these
conditions.
Figure 4: Transfer of BODIPY-LPS to unlabeled LPS micelles by rLBP or rPLTP-His. BODIPY-LPS (40 nM) was incubated in PBS alone (curve 1), with unlabeled LPS (4 µM, curve 2), with rPLTP-His (40 nM, curve 3), with rLBP (40 nM, curve 4), with unlabeled LPS (4 µM) and rPLTP-His (4 nM, curve 5, or 40 nM, curve 6), or with unlabeled LPS (4 µM) and rLBP (4 nM, curve 7, or 40 nM, curve 8). Fluorescence emission at 518 nM was measured over time.
[H]LPS incubated alone, with rLBP, with
rPLTP-His, or with R-HDL did not comigrate with the main band of R-HDL
seen with Coomassie staining (Fig. 5, lanes 1-5),
indicating that [
H]LPS does not spontaneously
bind to R-HDL under these conditions, as we have shown
previously(22) . However, upon incubation of
[
H]LPS with rLBP and R-HDL together, a portion of
the [
H]LPS migrated at the position of the main
Coomassie-stained band of R-HDL incubated with rLBP (Fig. 5, lanes 6-9). Similarly, incubation of
[
H]LPS with rPLTP-His and R-HDL together yielded
a radioactive band at the position of the main Coomassie-stained band
of R-HDL incubated with rPLTP-His (Fig. 5, lanes
10-13). The dramatic change in mobility of R-HDL caused by
rPLTP-His (compare lanes 5 and 13 of Fig. 5)
was time-dependent (not shown) and may reflect the ability of rPLTP-His
to mediate HDL conversion, a modification of HDL resulting in
populations of larger and smaller
particles(8, 9, 10) . Very similar
electrophoresis patterns were seen upon incubation of
[
H]LPS and R-HDL with rPLTP-His, with
plasma-derived PLTP, or with rPLTP medium, but not with control medium
(not shown). These results suggest that PLTP can transfer
[
H]LPS to R-HDL.
Figure 5:
Transfer [H]LPS to
R-HDL particles by rLBP or rPLTP-His. [
H]LPS (1
µg/ml) was incubated for 1 h at 37 °C in PBS alone (lane
1), with rLBP (10 µg/ml, lane 2), with rPLTP-His (13
µg/ml, lane 3), with R-HDL (100 µg/ml, lane
4), with R-HDL (100 µg/ml) and rLBP (0.1 µg/ml, lane
6; 1 µg/ml, lane 7; 10 µg/ml, lane 8),
or with R-HDL (100 µg/ml) and rPLTP-His (0.13 µg/ml, lane
10; 1.3 µg/ml, lane 11; 13 µg/ml, lane
12). The samples were subjected to native PAGE, and
[
H]LPS was revealed by fluorography. For
comparison, R-HDL (100 µg/ml) was incubated for 1 h at 37 °C in
PBS alone (lane 5), with rLBP (10 µg/ml, lane 9),
or with rPLTP-His (13 µg/ml, lane 13); the samples were
run in a separate gel and the gel was stained with Coomassie
Blue.
Figure 6: Transfer of BODIPY-LPS to R-HDL particles by rLBP or rPLTP-His. BODIPY-LPS (40 nM) was incubated in PBS alone (curve 1), with R-HDL (100 µg/ml, curve 2), with LBP (40 nM, curve 3), with rPLTP-His (40 nM, curve 4), with rLBP and R-HDL (curve 5), or with rPLTP-His and R-HDL (curve 6). Fluorescence emission at 518 nM was measured over time.
A 15-min incubation of
[H]LPS with sCD14 did not yield
[
H]LPS-sCD14 complexes (Fig. 7, lane
2). However, when the incubation was done in the presence of at
least 0.1 µg/ml rLBP, most of the [
H]LPS
comigrated with sCD14, suggesting complete transfer of
[
H]LPS to sCD14 by rLBP (Fig. 7, lanes
3-6), consistent with our previous findings(4) .
When [
H]LPS and sCD14 were incubated with
increasing concentrations of rPLTP-His (0.13-13 µg/ml), there
was no apparent formation of [
H]LPS-sCD14
complexes (Fig. 7, lanes 7-9). Similar
concentrations of rPLTP-His to those used here were able to transfer
[
H]LPS to R-HDL (Fig. 5), suggesting that
PLTP is able to transfer LPS, but unable to transfer LPS to CD14.
Figure 7:
Transfer of [H]LPS
to sCD14 by rLBP, but not by rPLTP-His. [
H]LPS (2
µg/ml) was incubated for 15 min at 37 °C in PBS alone (lane
1), with sCD14 (13 µg/ml, lane 2), with sCD14 (13
µg/ml) and rLBP (10 ng/ml, lane 3; 100 ng/ml, lane
4; 1 µg/ml, lane 5; 10 µg/ml, lane 6),
or with sCD14 (13 µg/ml) and rPLTP-His (130 ng/ml, lane 7;
1.3 µg/ml, lane 8; 13 µg/ml, lane 9). The
samples were subjected to native PAGE, and [
H]LPS
was revealed by fluorography.
PLTP also failed to transfer BODIPY-LPS to sCD14. Little change of fluorescence was seen when BODIPY-LPS was incubated alone or with rLBP, rPLTP-His, or sCD14 alone (Fig. 8, A and B, curves 1-4). However, incubation of BODIPY-LPS with CD14 and as little as 0.4 nM rLBP resulted in rapid dequenching of BODIPY-LPS (Fig. 8A, curves 5 and 6), reflecting rLBP-mediated binding of BODIPY-LPS to CD14(16) . In contrast, the change in fluorescence seen when BODIPY-LPS was incubated with rPLTP-His and CD14 together (Fig. 8B, curves 5 and 6) was the same as when BODIPY-LPS was incubated with the same concentrations of rPLTP-His alone (Fig. 8B, curves 3 and 4). Thus, concentrations of rPLTP which appear to transfer BODIPY-LPS to LPS acceptor micelles (Fig. 4B) or to R-HDL particles (Fig. 6) are unable to transfer LPS to CD14.
Figure 8: Transfer of BODIPY-LPS to sCD14 by rLBP (A), but not by rPLTP-His (B). A, BODIPY-LPS (40 nM) was incubated in PBS alone (curve 1), with sCD14 (40 nM, curve 2), with rLBP (0.4 nM, curve 3; 4 nM, curve 4), or with sCD14 (40 nM) and rLBP (0.4 nM, curve 5; 4 nM, curve 6). Fluorescence emission at 518 nM was measured over time. B, BODIPY-LPS (40 nM) was incubated in PBS alone (curve 1), with sCD14 (40 nM, curve 2), with rPLTP-His (40 nM, curve 3; 400 nM, curve 4), or with sCD14 (40 nM) and rPLTP-His (40 nM, curve 5; 400 nM, curve 6). Fluorescence emission at 518 nM was measured over time.
We measured adhesive responses of neutrophils to LPS to confirm the inability of rPLTP-His to transfer LPS to CD14. Neutrophils do not adhere to fibrinogen-coated plates in response to LPS alone, but do adhere in response to LPS with LBP(2, 4) . This response depends on mCD14, since it can be inhibited with monoclonal antibodies to CD14 (4) . We therefore tested the ability of rPLTP-His to mediate this mCD14-dependent response by incubating neutrophils with a fixed dose of LPS and increasing amounts of rLBP or rPLTP-His (Fig. 9). Addition of as little as 1 ng/ml rLBP allowed adhesion of neutrophils, whereas addition of rPLTP-His did not enhance the adhesive response at any concentration tested. This result, and the previous experiments ( Fig. 7and Fig. 8), strongly suggest that PLTP is unable to transfer LPS to CD14.
Figure 9:
rPLTP-His does not mediate responses of
neutrophils to LPS. Neutrophils were incubated for 10 min at 37 °C
in buffer alone (-
), with rLBP
(
-
), with rPLTP-His
(
-
), with LPS (10 ng/ml)
(
-
), with LPS and rLBP
(
-
), or with LPS and rPLTP-His
(
-
). Adhesion of neutrophils to
fibrinogen-coated plates was assessed as described under
``Materials and Methods.''
Figure 10:
rPLTP-His inhibits IL-6 production in
whole blood in response to LPS. 50% (v/v) whole blood was incubated for
5 h with the indicated concentrations of LPS in the absence
(]-
) or presence of rPLTP-His (10
µg/ml) (
-
). Since rPLTP-His was added in
low-pH buffer, LPS was added with the equivalent volume of this buffer
as a control (
-
).
Here we have shown that PLTP, a plasma protein known to
transfer phospholipids (6) and mediate HDL
conversion(8, 9, 10) , inhibits cellular
responses to LPS. The observed inhibition was due to the action of PLTP
on LPS, rather than on cells, because neutralization of LPS by PLTP was
dependent on the time of incubation of LPS with PLTP (Fig. 1A), and rPLTP-His had no effect on tumor
necrosis factor -induced adhesion of neutrophils (not shown). The
ability of PLTP to cause dequenching of BODIPY-LPS (Fig. 3)
provides additional evidence for a physical interaction of PLTP with
LPS and suggests that PLTP binds LPS. The binding of BODIPY-LPS by a
protein would be expected to cause dequenching, due to the physical
separation of BODIPY molecules, and the effect of PLTP on the
fluorescence of BODIPY-LPS was similar to the effect of LBP (Fig. 3), a protein known to bind
LPS(19, 20, 21) .
The above results suggest that PLTP may neutralize LPS by binding it. This mechanism is consistent with the previous identification of several proteins and peptides which bind and neutralize LPS. These include bactericidal/permeability-increasing protein (BPI)(28, 29) , a neutrophil granule protein with sequence similarity to LBP (44%) (3) and PLTP (26%)(5) ; Polymixin B, an acylated cationic peptide with antimicrobial activity for Gram-negative bacteria(30, 31) ; endotoxin neutralizing protein, a protein from Limulus which neutralizes LPS in vitro(32) ; and CAP-18, a cationic protein of neutrophil granules(33) . Like PLTP, all these proteins appear to neutralize LPS simply by binding it and preventing its recognition by CD14 or by other cellular factors necessary for responses to LPS.
The stoichiometry and affinity of binding of PLTP to LPS are not evident from our studies and may depend on the buffer conditions. A greater rise in the fluorescence of BODIPY-LPS was caused by PLTP and LBP with EDTA present (Fig. 3A) than without EDTA and with divalent cations (Fig. 3B). EDTA does not appear to change the intrinsic fluorescence of BODIPY-LPS, because fluorescence of BODIPY-LPS alone was unaffected by EDTA (Fig. 3, A and B), and the fluorescence of complexes of BODIPY-LPS and CD14 was the same in PBS and PD-EDTA (not shown). EDTA prevents the binding of divalent cations to LPS(34) , thereby weakening lateral interactions between LPS molecules(35) . By destabilizing interactions between LPS molecules, EDTA may increase the affinity of LBP and PLTP for LPS or it may allow the formation of BODIPY-LPS-PLTP or BODIPY-LPS-LBP complexes with low LPS:protein ratios, which would have greater fluorescence due to the lower aggregation state.
We also present evidence here that PLTP transfers
LPS, either between micelles of LPS or to R-HDL particles. The
incubation of micelles of BODIPY-LPS with unlabeled LPS micelles or
with R-HDL particles resulted in rapid dequenching only in the presence
of PLTP or LBP ( Fig. 4and 6), a finding consistent with
transfer of BODIPY-LPS molecules. Alternatively, fluorescence
dequenching might result from fusion of BODIPY-LPS micelles with LPS
micelles or with R-HDL particles. The PLTP-mediated incorporation of
[H]LPS into R-HDL did not appear to change the
molecular weight of R-HDL particles (Fig. 5, compare lanes
12 and 13), making fusion of an entire LPS micelle with a
single R-HDL particle an unlikely mechanism. However, we cannot rule
out the possibility that PLTP and LBP mediate the fusion of small
aggregates of LPS with R-HDL particles, since this mechanism would have
similar consequences as the transfer of LPS.
The ability of PLTP to transfer LPS, a membrane lipid of Gram-negative bacteria, is consistent with its ability to transfer other amphipathic molecules. PLTP has been found to transfer a variety of glycerophospholipids as well as sphingomyelin and thus appears to have little specificity for the fatty acyl composition or head group of phospholipids(36) . LPS is a much larger molecule than phospholipids, having six or seven acyl chains and at least four carbohydrate groups (37) . Nonetheless, the overall amphipathic nature of the molecules is similar, and the solubility of LPS in aqueous environments resembles that of glycerophospholipids(37) . Our results suggest that LPS is sufficiently similar to phospholipids that it is recognized and transferred by PLTP.
PLTP and LBP share the ability to transfer LPS to R-HDL ( Fig. 5and 6). The LBP-mediated transfer of LPS to R-HDL particles has been shown to result in the neutralization of LPS(2) . Because PLTP also appears to transfer LPS to R-HDL, it may also neutralize LPS by transferring it and may have a role in the transfer of LPS to lipoproteins in vivo. In our studies, PLTP neutralized LPS on its own ( Fig. 1and 2), and the rate of neutralization was not increased by adding R-HDL (not shown); therefore, we could not directly demonstrate whether PLTP could neutralize LPS by transfer to R-HDL particles. In principle, however, PLTP may neutralize LPS through two distinct mechanisms via direct interaction or by transferring LPS to lipoprotein particles. Either or both of these mechanisms may account for the finding that adding PLTP to whole blood caused a reduced sensitivity of cytokine release in response to LPS (Fig. 10).
PLTP is unable to transfer LPS to CD14. We have previously studied the transfer of LPS to CD14 by LBP using native gel electrophoresis (4) and fluorescence dequenching of BODIPY-LPS(16) ; using these techniques, we were unable to detect transfer of LPS to CD14 by PLTP ( Fig. 7and Fig. 8). PLTP was also unable to mediate adhesion of neutrophils to fibrinogen in response to LPS (Fig. 9). This cellular response is dependent on mCD14(14) , and the ability of LBP to mediate this response most likely reflects its ability to transfer LPS to CD14. Our results suggest that while LBP is not unique in its ability to transfer LPS, it may be unique in its ability to transfer LPS to CD14. PLTP is the first protein described which is able to transfer LPS, but which does not transfer LPS to CD14. Our results support the possibility that unique sequences in LBP allow it to interact with CD14, as suggested by the finding that the N-terminal portion of LBP binds LPS but does not transfer LPS to CD14(38, 39) . Further studies may identify the putative sequences in LBP which allow transfer of LPS to CD14.
PLTP is part of a gene family that also includes LBP, BPI, and CETP. The abilities of LBP and BPI to interact with LPS are well established, and our results show that PLTP shares this property. CETP may also interact with LPS, inasmuch as the transfer of cholesteryl esters by plasma from mice transgenic for human CETP is inhibited by LPS(40) . Levels of LBP and BPI may rise during infection, as LBP is an acute phase reactant (20) and BPI is released by activated neutrophils(28) . On the other hand, PLTP mRNA levels are decreased by injection of LPS into mice(41) , and CETP transcription and protein levels decline upon injection of LPS in mice transgenic for human CETP(40) . The role of these changes in modulating responses to LPS in different disease states and sites of infection may be a fruitful area of study.