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J Biol Chem, Vol. 274, Issue 48, 34116-34122, November 26, 1999
From the Departments of When bacterial lipopolysaccharide (LPS) enters
the bloodstream, it is thought to have two general fates. If LPS binds
to circulating leukocytes, it triggers innate host defense mechanisms
and often elicits toxic reactions. If instead LPS binds to plasma
lipoproteins, its bioactivity is largely neutralized. This study shows
that lipoproteins can also take up LPS that has first bound to
leukocytes. When monocytes were loaded with [3H]LPS
and then incubated in plasma, they released over 70% of the
cell-associated [3H]LPS into lipoproteins (predominantly
high density lipoprotein), whereas in serum-free medium the
[3H]LPS remained tightly associated with the cells. The
transfer reaction could be reproduced in the presence of pure native
lipoproteins or reconstituted high density lipoprotein. Plasma
immunodepletion experiments and experiments using recombinant LPS
transfer proteins revealed that soluble CD14 significantly enhances LPS
release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell
surface can be released. Lipoprotein-mediated LPS release was
accompanied by a reduction in several cellular responses to the LPS,
suggesting that the movement of LPS from leukocytes into lipoproteins
may attenuate host responses to LPS in vivo.
Gram-negative bacterial lipopolysaccharide
(LPS)1 (endotoxin) is one of
the most potent and ubiquitous of the known bacterial signal molecules.
Animals have sensitive mechanisms for recognizing the presence of LPS
in tissues. Monocytes, macrophages, and neutrophils, which express the
LPS binding receptor, CD14 (1, 2), and its signal transducer, Toll-like
receptor-4 (3), are particularly sensitive to LPS (4). They respond by
producing inflammatory mediators that amplify and diversify the LPS
signal, triggering host defenses that wall off and destroy invading
bacteria. For reasons that are not entirely clear, however, the
response to LPS can also be harmful, resulting in severe sepsis, septic
shock, and even death. Mechanisms that regulate responses to LPS are therefore likely to be very important for the host.
HDL is the most abundant of the lipoproteins in human plasma and
interstitial fluids. It can remove both phospholipids and unesterified
cholesterol from cells (reviewed in Ref. 5), and HDL-mediated cellular
cholesterol efflux and the subsequent delivery of HDL-cholesterol to
the liver (reverse cholesterol transport) are thought to help protect
animals from atherosclerosis. This study addresses another facet of the
HDL particle: its ability to bind LPS and neutralize its biological
activity. There is abundant evidence that HDL and other plasma
lipoproteins play an important role in controlling host responses to
LPS. Numerous studies have shown that complexes of LPS with HDL and
other lipoproteins have little or no stimulatory activity, either
in vitro or in vivo (6-9), and there is now
strong evidence that HDL and other lipoproteins can neutralize
endotoxin in vivo (10-17).
Two plasma lipid transfer proteins, LPS-binding protein (LBP) (18, 19)
and phospholipid transfer protein (PLTP) (20), promote the binding of
purified LPS to lipoproteins, whereas only LBP can facilitate LPS
binding to CD14 on cell membranes (mCD14) or soluble CD14 (sCD14) in
plasma (1, 21, 22). sCD14 can rapidly transfer LPS to mCD14 on cells
(23), and it also facilitates the activation of cells that do not
express mCD14 (24). Although sCD14 can also transfer LPS to HDL (19),
it has been said to contribute very little to the movement of LPS to
lipoproteins in whole plasma (25).
In this study, we sought to determine whether HDL and plasma LPS
transfer proteins could remove LPS from host cells. We found that HDL
facilitates the release of cell-bound LPS, that sCD14 enhances this
release, and that the removal of LPS from the cells attenuates
proinflammatory responses. This new pathway of LPS traffic to
lipoproteins may be important for regulating host responses to
Gram-negative bacteria. These findings also raise the possibility that
efflux of microbial ligands from macrophages to HDL contributes to the
anti-inflammatory potency of HDL in processes such as atherosclerosis.
Cells and Reagents--
Human monocytic THP-1 cells were
cultured as described previously (26). CD14 expression was induced
either by culturing the cells in 1,25-dihyroxyvitamin D3
(26, 27) or by transforming the cells with human CD14 cDNA (23).
Peripheral blood mononuclear cells (PBMC) were separated from
heparinized blood of normal human donors by centrifugation over
Histopaque 1077 (Sigma) (28). Plasma was obtained from normal human
volunteers without the use of anticoagulants as described (29).
Briefly, venous blood was mixed immediately with streptokinase
(Calbiochem) (150 units/ml blood), which accelerates fibrinolysis, and
platelet-free plasma was separated by centrifugation. Recombinant human
LPS-binding protein (rLBP) was partially purified on SP-Sepharose (30)
from serum-free culture supernatants overlying Chinese hamster ovary cells (a gift of P. Tobias, Scripps Research Institute, La Jolla, CA)
that expressed the protein (23). Purified histidine-tagged LBP
(rLBP-His), expressed in baculovirus-infected insect cells, was also
provided by P. Tobias. Purified recombinant human soluble CD141-356 (rsCD14) was provided by R. Thieringer and
S. D. Wright (Merck). RPMI 1640 medium, Cellgro Complete
serum-free medium, and G418 were from Mediatech (Herndon, VA). All
other reagents were from Sigma unless otherwise stated.
Lipoproteins--
Native lipoproteins were isolated from plasma
by sequential ultracentrifugal flotation in potassium bromide (KBr) at
densities of 1.019 (VLDL), 1.063 (LDL), and 1.21 (HDL) g/ml (31). For some experiments, the total lipoprotein fraction of plasma (0-1.21 g/ml) was isolated. The lipoproteins were concentrated by
recentrifugation and dialyzed thoroughly against cold 0.9% NaCl
containing 0.25 mM EDTA (pH 8). Lyophylized R-HDL (32), a
gift of Dr. P. Lerch (Swiss Red Cross Blood Transfusion Service, Bern,
Switzerland), was reconstituted in distilled water and dialyzed for
24 h (2-3 changes) in the above buffer to remove free cholate.
Apolipoprotein, Cholesterol, and Phospholipid
Assays--
Apolipoproteins A-I and B were measured by turbidometric
assay kits from Sigma. Total (esterified and unesterified) cholesterol and choline-containing phospholipids were measured using kits from
Sigma and Wako Chemical Co. (Richmond, VA), respectively.
Immunodepletion of Plasma LPS Transfer Proteins--
Anti-human
LBP monoclonal antibody 18G4 (a gift of P. Tobias, Scripps Research
Institute) or nonimmune mouse IgG was covalently bound to Avidchrom
Hydrazide F Gel beads (Unisyn Technologies, Hopkinton, MA) according to
the manufacturer's instructions. Fresh normal human plasma or serum
was mixed with sedimented mAb-conjugated beads on a rocking platform
overnight at 0-4 °C. The beads were removed by centrifugation, and
LBP was measured in the depleted and control sera by ELISA; the capture
antibody (mAb IE8), the detection antibody (biotinylated mAb 18G4), and
the LBP standard solution (rLBP-His) were provided by P. Tobias. Plasma
LBP activity was measured by the ability of whole undiluted plasma to
promote binding of [3H]LPS to mCD14 on THP-1 cells;
[3H]LPS (2.5 or 10 ng) was incubated with plasma (50 µl) and cells (3.5 × 105) for 5 or 10 min at
37 °C, the cells were washed with cold PBS, and cell-associated
[3H]LPS was measured (see below). Because control
antibody beads or unconjugated beads also removed up to 90% of the LBP
due to nonspecific adhesion, the precolumn serum was used as a control. The beads removed only 20% of PLTP activity and 54% of the sCD14.
sCD14 was removed by incubating 500 µl of plasma with anti-CD14 mAb
60bca (20 µg) for 1 h. The mAb was then captured and removed by
adding streptavidin-coated magnetic beads (Dynal, Lake Success, NY)
that were saturated with biotin-SP-conjugated rabbit anti-mouse IgG (H + L) (Jackson Immunoresearch Laboratories, West Grove, PA). The plasma
was mixed with the beads on a rocking platform at 0-4 °C (three
times with 1.5 mg of beads for 1 h each and once with 1.5 mg of
beads for 16 h), and the beads were removed by brief
centrifugation. Control plasma was produced in the same way, except
that a nonimmune control antibody was used (MOPC-21, IgG1
PLTP activity was neutralized by incubating plasma with polyclonal
rabbit anti-human PLTP. Nonimmune rabbit IgG was added as a control.
Plasma PLTP activity was measured as described (33).
LPS Preparation, Cell Binding, and
Release--
Escherichia coli LCD25 [3H]LPS
(1.5 × 106 dpm/µg) was biosynthetically labeled and
isolated as described (34). BODIPY-LPS was prepared from E. coli LCD25 LPS as described (28). For cell binding, the LPS was
diluted in HNEB (20 mM HEPES buffer, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 0.3 mg/ml bovine serum
albumin) (23) and mixed with rsCD14 (25 µg protein/µg LPS) and rLBP
(0.1 µg protein/µg LPS), incubated for 10 min at 37 °C, and
diluted with serum-free medium (SFM) (Cellgro Complete serum-free
medium, 20 mM HEPES, pH 7.4, and 0.3 mg/ml bovine serum
albumin). THP-1 cells (7 × 106/ml) or PBMC
(15-25 × 106/ml) were warmed to 37 °C, the
LPS-sCD14-LBP mixtures were added (50 ng LPS/ml final concentration),
and the incubation was continued for 5 min at 37 °C. Unbound LPS was
then removed by centrifugation, and the cells were washed three times
with ice-cold SFM. The cells were resuspended either in undiluted
plasma containing 20 mM HEPES, pH 7.4, or in SFM with or
without lipoproteins or LPS transfer proteins. 50 or 100 µl of cell
suspension was added to 1.5-ml microcentrifuge tubes and incubated at
37 °C with gentle mixing to keep cells in suspension. The
incubations were stopped with 0.3 ml of cold PBS, the cells were
removed by centrifugation, and the radioactivity in the cells and
supernatants was counted to measure bound and released
[3H]LPS. The mean fluorescence intensity of BODIPY-LPS
that remained associated with the cells was measured by flow cytometry
(23). In some experiments, surface-exposed and internalized fractions of cell-associated LPS were measured either by proteolytic release of
surface-bound LPS by incubating the cells for 30 min in 0.02% proteinase K at 0-4 °C or by quenching of surface-exposed
BODIPY-LPS with trypan blue as described (23, 28). Under the conditions of our assays, less than 2% of the cell-associated
[3H]LPS was deacylated by cellular acyloxyacyl hydrolase
(35) (not shown).
Cell counting and trypan blue permeability tests performed after the
incubations revealed that [3H]LPS release was not due to
cell loss or alterations in plasma membrane permeability. Control and
immunodepleted plasma samples were assayed in the absence or presence
of 10 mM EDTA to inhibit complement fixation (36)
(e.g. from anti-PLTP-PLTP immune complexes). A significant
degree of membrane permeability (>10% permeable cells) was observed
only in plasma samples that contained anti-PLTP or high concentrations
of rsCD14; cell permeability induced by these reagents was completely
inhibited by EDTA or by removal of plasma. EDTA did not alter the
percentage of cell-associated LPS released into control or
sCD14-depleted plasma.
The [3H]LPS distribution among the major plasma
lipoprotein classes was measured by ultracentrifugal flotation of
undiluted plasma supernatants as described above or by size exclusion
chromotography on a Superose 6 HR 10/30 column (Amersham Pharmacia
Biotech) (37).
Cell Stimulation Assays--
Cells were loaded with LPS (0.03-3
ng/ml) and washed as described above. The cells were then incubated in
100 µl of SFM (4 × 106 THP-1 cells/ml) or RPMI 1640 medium containing 10 mM HEPES, pH 7.4, and 0.1 mg of bovine
serum albumin/ml (7-12 × 106 PBMC/ml) in
microcentrifuge tubes or 96-well culture plates, respectively. In some
incubations, recombinant human IL-1 Human plasma rapidly removes LPS from monocytes. LPS
binds rapidly to mCD14 on monocytes and THP-1 cells; maximal binding occurs within a few minutes. In contrast, bound LPS dissociates from
the cells very slowly in the absence of serum, even when the cells are
treated with inhibitors that prevent LPS internalization (23). In the
experiments shown in Fig. 1,
CD14-transfected THP-1 cells were loaded with [3H]LPS for
5 min at 37 °C, washed to remove the unbound LPS, and then
reincubated in either SFM or human plasma. As expected,
[3H]LPS bound to the cells rapidly and then dissociated
extremely slowly from cells that were suspended in SFM. When plasma was added after the cells were loaded with LPS, in contrast, the cells rapidly released most of the LPS they had bound. Plasma-mediated LPS
release was not due to cell loss or membrane permeability, as trypan
blue exclusion by the cells was not affected (not shown).
Cell-derived LPS Binds to Plasma Lipoproteins--
We next asked
whether the released [3H]LPS was transferred to plasma
lipoproteins. After the [3H]LPS-loaded cells had been
incubated with plasma for 30 min, the major lipoprotein classes (VLDL,
LDL, and HDL) in the culture supernatants were separated by
ultracentrifugation in KBr at different densities (d) to
isolate VLDL, IDL, and chylomicrons (d
Having shown that at least 80% of the released [3H]LPS
was bound to lipoproteins, we also separated the lipoproteins according to their particle sizes on a Superose 6 gel filtration column (Fig.
2); most of the cell-derived
[3H]LPS co-eluted with HDL and LDL, the majority being in
the HDL fractions. Whether the distribution of lipoprotein-bound
[3H]LPS was measured by centrifugal flotation (Table I)
or gel filtration (Fig. 2), it correlated closely with phospholipid
content but not with total cholesterol. Taken together, these
experiments showed that HDL was the dominant acceptor for cell-bound
LPS in normal plasma (HDL > LDL Purified Plasma Lipoproteins Promote the Release of Cell-associated
LPS--
We next studied LPS release from THP-1 cells into serum-free
medium to which pure lipoproteins were added, with or without added
recombinant LPS transfer proteins. The results show that nHDL (Fig.
3, A-C) or R-HDL (Fig. 3,
D-F) promoted LPS release from the cells without a
requirement for soluble LPS transfer proteins. R-HDL promoted more
rapid LPS release than did nHDL, in keeping with the ability of R-HDL
to rapidly adsorb free LPS (32). In experiments not shown, nLDL
promoted LPS release with kinetics that were similar to those of nHDL.
Adding rsCD14 to nHDL (Fig. 3A) or R-HDL (Fig.
3D) accelerated the release of cell-associated LPS. Testing
different concentrations of sCD14 showed that maximal release was
obtained at approximately 5 µg of sCD14/ml (not shown). rLBP also
accelerated the release of cell-associated LPS in the presence of R-HDL
(Fig. 3E), with maximal release at 0.03 µg of rLBP/ml,
whereas the same concentration of rLBP had no effect in the presence of
nHDL (not shown). However, when much higher concentrations (10 µg of
rLBP-His/ml) were added, the LBP slightly enhanced LPS release from the
cells in the presence of nHDL (Fig. 3B). The low activity of
the rLBP-His in this assay was not due to its inability to transfer
LPS; 1 µg of rLBP-His/ml completely restored the ability of
LBP-depleted plasma to promote [3H]LPS binding to mCD14
on cells, and the activities of the rLBP and rLBP-His were similar (not
shown). PLTP had no effect in the presence of either lipoprotein (Fig.
3, C and F) at concentrations (up to 20 µg
rPLTP/ml) that were many fold higher than normal plasma PLTP
concentrations. The lack of effect of rPLTP was not due to a lack of
LPS transfer activity. In experiments not shown, less than 1 µg of
rPLTP/ml transferred 80% of free [3H]LPS (200 ng/ml) to
native lipoproteins, whereas rPTLP was unable to transfer much smaller
amounts (~20 ng/ml) of cell-bound [3H]LPS to HDL (Fig.
3, C and F) or LDL (not shown). rPLTP did not contribute to the release of LPS when it was added together with rsCD14
or rLBP (not shown).
No significant release of cell-associated LPS occurred in the presence
of rLBP or rPLTP alone (Fig. 3, B, C, E, and F).
In contrast, when sCD14 was added alone (Fig. 3, A and
D), the cells released 20-30% of their LPS within 7 min
and maintained the same percentage of released LPS throughout the time
course of the incubation. Maximal LPS release did not occur unless
lipoproteins were added. These data suggest that an equilibrium is
rapidly established between cell-associated LPS and soluble LPS-sCD14
complexes, whereas the addition of a stronger LPS acceptor (HDL) shifts
the equilibrium toward the lipoprotein particles and allows the bulk of
the LPS to be removed from the cells.
sCD14 Enhances the Release of LPS into Human Plasma--
To test
whether normal plasma levels of the three LPS transfer proteins are
important for accelerating LPS release from cells into whole plasma, we
immunodepleted or neutralized LBP, PLTP, or sCD14 using specific
antibodies. Immunodepletion removed 97 ± 4 (n = 8) and 99.6 ± 0.3% (n = 5) of the immunoreactive
plasma sCD14 and LBP, respectively. As expected, LPS binding assays
showed a marked reduction in the ability of LBP-depleted plasma to
promote [3H]LPS binding to THP-1 cells; 49.1 ± 0.1% of the added [3H]LPS bound to the cells in control
plasma, whereas 4.4 ± 2.8% bound in LBP-depleted plasma
(mean ± S.D.; n = 3). Anti-PLTP neutralized 97 ± 1% (n = 3) of the plasma PLTP activity.
As shown in Fig. 4, reducing sCD14 levels
significantly decreased the release of cell-associated
[3H]LPS into plasma, whereas reducing LBP or PLTP levels
had no effect. Neutralization of PLTP in LBP-depleted plasma also had no effect on LPS release from cells (not shown). The amount of cell-associated [3H]LPS released into sCD14-depleted
plasma was the same as that released into serum-free medium to which
were added approximately normal concentrations of plasma lipoproteins
(isolated by KBr density flotation (d < 1.21 g/ml));
the isolated lipoprotein fraction was essentially devoid of sCD14, LBP,
and PLTP (not shown). Normal activity was restored to sCD14-depleted
plasma by adding physiologic concentrations (2 µg/ml) of rsCD14 (Fig.
4), whereas adding rPLTP (10 µg/ml) or rLBP (0.5 µg/ml) had no
effect, and adding rLBP-His (10 µg/ml) only slightly increased LPS
transfer.
In the Presence of Lipoproteins, Monocytes Release Almost All
Cell-Surface LPS--
As noted above, the fraction of the total
cell-associated LPS that was released from the cells in the presence of
lipoproteins reached a plateau at approximately 70-80%. We used two
LPS internalization assays (23, 28) to determine the cellular location
of the remaining 20-30% of the LPS. In the experiment shown in Fig.
5, the release of cell-associated LPS was
measured in normal human monocytes (mononuclear cells). After various
incubation times in the presence of R-HDL, the cells were washed and
incubated with 0.02% proteinase K on ice to remove LPS from mCD14 or
other protease-sensitive LPS-binding proteins on the cell surface.
Before the cells were exposed to R-HDL (Fig. 5A, time 0),
the protease released 78% of the total cell-associated
[3H]LPS. After 60 min in the presence of R-HDL + rLBP,
the cells released 73% of the [3H]LPS into the medium
(not shown), and protease treatment released only an additional 2% of
the original cell-associated [3H]LPS (Fig.
5A). The results suggest that R-HDL promoted the release of
nearly all of the surface-bound (protease-sensitive) LPS from the
cells, whereas the remaining cell-associated LPS was protease-resistant (Fig. 5B). Because surface-exposed LPS that is inserted into
the membrane or bound to a protease-resistant protein would be counted with the internalized LPS in this assay, we measured cell-associated BODIPY-LPS by flow cytometry before and after quenching the
fluorescence of the surface-exposed BODIPY-LPS with trypan blue. Before
rewarming the cells (Fig. 5C, 0-4 °C), the surface and
internal fractions of LPS were 80 and 20%, respectively. After
incubation with R-HDL, surface LPS decreased dramatically, whereas
there was little change in internalized LPS. Similar results were
obtained using normal human monocytes (not shown). These data suggest
that virtually all of the cell-surface LPS is rapidly released in the
presence of lipoprotein particles, leaving only internalized LPS.
HDL Attenuates Signal Responses in LPS-loaded Cells--
The cells
were incubated for 5 min at 37 °C with LPS concentrations that were
sufficient to stimulate cytokine responses. The unbound LPS was removed
by washing, the cells were incubated in fresh serum-free medium with or
without R-HDL or nHDL, and cytokines released by the cells were
measured. IL-8, which is produced rapidly in response to LPS (27), was
measured in the medium after incubation for 2 h, whereas the cells
were incubated for 16 h before measuring cytokines that are
produced more slowly (TNF- Although numerous studies have shown that HDL and other
lipoproteins can bind and neutralize the biological activity of free LPS, lipoprotein-mediated removal of LPS from cell membranes has not
been previously described. We found not only that LPS can leave the
monocyte surface and bind lipoproteins, but also that exposing
LPS-loaded cells to HDL significantly reduces the production of several
cytokines (TNF- Our data suggest that prolonged contact of the cells with LPS must be
maintained in order to produce LPS signals of maximal strength and
duration. This hypothesis is supported by other studies. For example,
prolonged expression of LPS-induced inflammatory genes (TNF- Because the kinetics of LPS binding to monocytes in whole plasma are
similar to those that occur in plasma-free medium containing rLBP and
rsCD14 (not shown), this suggests that a large fraction (up to 50%) of
the plasma LPS transiently associates with the cells before it moves
into lipoproteins. The degree of cell stimulation may thus be related
to the persistence of the interaction of CD14-bound LPS with signaling
receptors (Toll-like receptor-4) on the cell surface. On the other
hand, if LPS interactions with membrane lipids and endocytosis are
important for signaling (43), then the degree of cell stimulation could
be related to flow of LPS into these sites.
Although plasma HDL was the dominant acceptor of cell-bound LPS in our
experiments, LDL also bound a significant proportion of the released
LPS. Many lipoproteins (VLDL, LDL, and chylomicrons) and lipid
emulsions can bind and neutralize LPS (12, 44-46). Some studies have
found that LDL is able to neutralize LPS better than HDL (47), but when
several lipoprotein classes were compared according to their
phospholipid content, LPS neutralizing ability was similar in all
lipoprotein classes (14). Moreover, increasing the phospholipid to
apolipoprotein ratios in reconstituted HDL particles increased their
ability to neutralize LPS (15, 41). We found that LPS movement into
lipoproteins correlated with their phospholipid content. Thus,
phospholipids may be important for both release and neutralization of
cell-associated LPS. Phospholipids are the primary constituents of the
HDL particle surface, and, in most individuals, the large numbers of
HDL particles present in plasma provide the greatest effective surface
area of any of the circulating lipoproteins. We therefore hypothesize
that the abundance of phospholipid on HDL particle surfaces contributes to its superior ability to bind released LPS.
The mechanisms involved in transferring LPS from cells to lipoproteins
may be complex. Numerous studies have shown that cholesterol efflux
from cells to lipoproteins can occur by both
receptor-dependent and receptor-independent mechanisms
(reviewed in Refs. 5 and 48). Class B scavenger receptors (SR-BI (49)
and CD36 (50)) bind native HDL and LDL as well as modified
lipoproteins, and recent studies show that SR-BI promotes the efflux of
unesterified cholesterol from cells to HDL or LDL (51, 52). Because
human monocytes express both SR-BI and CD36 (50, 53), we are
investigating the roles of these receptors in cellular "efflux" of
LPS. Just as cholesterol efflux from cell membranes is thought to occur both by receptor-mediated and non-receptor-mediated processes (48), it
seems likely that LPS efflux may also occur by both kinds of mechanisms.
We found that sCD14 enhanced the release of cell-bound LPS in the
presence of isolated nHDL or plasma, whereas LBP and PLTP had little or
no effect. Although sCD14 accelerated LPS transfer from cells to both
R-HDL and nHDL, we found unexpectedly that rLBP strongly enhanced LPS
release in the presence of R-HDL. LBP (18, 54) is normally found
attached to native HDL particles in plasma, but it is lost when HDL is
isolated using KBr, and we were unable to detect it by ELISA in our
preparations of nHDL (data not shown). Although traces of contaminating
LBP might contribute to the ability of nHDL to release cell-surface LPS
(and thus to the inability of rLBP to increase LPS release much above
that seen with nHDL alone), this explanation thus seems unlikely.
Our results suggest that the interactions between LPS, lipoproteins,
plasma transfer proteins, and leukocytes may be more complex than has
previously been suspected (Fig. 6).
Whereas both purified and native LPS were known to bind to leukocytes
and lipoproteins, these were generally thought to be irreversible
interactions that either inactivated the LPS (lipoprotein binding) or
led to cellular activation (cell binding and internalization). It now
seems that the LPS-leukocyte interaction is probably quite dynamic,
with plasma transfer proteins facilitating both the binding and release of LPS from cells when lipoproteins are present. It is also possible that before LPS-lipoprotein complexes are cleared from the circulation, the LPS can move from lipoproteins to cells; the ability of the lipoproteins to sequester LPS effectively may be due to the large size
of the lipoprotein "acceptor sink." The concentrations of circulating lipoproteins and LPS transfer proteins may thus be very
important determinants of the fate of LPS molecules in plasma.
Plasma Lipoproteins Promote the Release of Bacterial
Lipopolysaccharide from the Monocyte Cell Surface*
§,
Internal Medicine and
Microbiology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235-9113 and the ¶ Department of Medicine and
Northwest Lipid Research Laboratories, University of Washington,
Seattle, Washington 98103-9103
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
; Sigma).
Plasma sCD14 was measured by ELISA (R & D Systems, Minneapolis, MN).
The low sCD14 levels in the immunodepleted plasma were not due
interference by residual antibody, because the addition of 60bca to
plasma samples before and during ELISA had no inhibitory effect on the assay.
(final concentration, 5-10
ng/ml) (Pharmingen, Los Angeles, CA) was added. IL-8 was measured in
the culture supernatants after 2 h, and TNF-
, IL-1, and IL-6
were measured after incubating the cells in a 5% CO2
atmosphere 16-20 h. The cytokines were measured by ELISA using DuoSet
kits or match antibody pairs from Genzyme, Cambridge, MA (IL-8,
IL-1, and IL-6) or Pharmingen (TNF-).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Whole plasma removes LPS from cells.
CD14-transfected THP-1 cells were loaded with [3H]LPS,
washed, and incubated with SFM or plasma from three normal human donors
as described under "Experimental Procedures." The y axis
shows the percentage of total cell-associated [3H]LPS
(3000-3300 dpm) that remained associated with the cells.
1.019 g/ml),
LDL (1.019 < d 1.063 g/ml), and HDL
(1.063 < d 1.21 g/ml). The recoveries of
[3H]LPS, total cholesterol, and phospholipids were
74 ± 9, 86 ± 3, and 87 ± 7%, respectively (mean ± S.D.; n = 4). After correcting for recovery of
lipoprotein cholesterol, 80% of the released LPS was found in
lipoproteins (the majority was in HDL), whereas only 5.6% was in
nonlipoprotein fractions (d > 1.21 g/ml), and 14% was
not accounted for (Table I). In control
experiments, free [3H]LPS did not float at any KBr
density in the absence of plasma, even when the LPS was prebound to
sCD14 or LBP or released from cells by protease treatment.
Distribution of released LPS among human plasma lipoproteins separated
by ultracentrifugal flotation
VLDL), and the distribution
among the fractions correlated with the phospholipid content of the
lipoproteins. In experiments not shown, HDL was also the dominant
acceptor when free [3H]LPS was added to plasma.
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Fig. 2.
Chromatographic separation of plasma
[3H]LPS released by cells. Whole human plasma was
incubated with [3H]LPS-loaded THP-1 cells for 30 min as
in Fig. 1. 200 µl of the resulting plasma was separated on a Superose
6 column, and the fractions were assayed for phospholipids
(µg/fraction), total cholesterol (µg/fraction), and
[3H]LPS (dpm divided by 10) per fraction. Recoveries of
phospholipids, cholesterol, and [3H]LPS were 92, 91, and
80%, respectively. HDL peak fractions contained apoA-I but not apoB,
whereas LDL peak fractions contained apoB but not apoA-I (not shown).
The experiment was repeated with plasma from a different donor with
similar results.
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Fig. 3.
Pure HDL particles promote the release of
cell-associated LPS. [3H]LPS-loaded THP-1 cells were
incubated with SFM with or without nHDL (1 mg of apoA-I/ml)
(A-C) or R-HDL (0.1 mg of apoA-I/ml) (D-F) with
or without rsCD14 (5 µg/ml) (A and D), rLBP-His
(10 µg/ml) (B), rLBP (0.06 µg/ml) (E), or
rPLTP (C and F) for the indicated times and
analyzed as in Fig. 1. Each experiment was performed at least three
times with similar results.
View larger version (18K):
[in a new window]
Fig. 4.
Immunodepletion of sCD14 from normal plasma
reduces the release of cell-associated LPS.
[3H]LPS-loaded THP-1 cells were incubated for 30 min at
37 °C with control plasma or plasma immunodepleted of LBP
(LBP 
), PLTP (PLTP), or sCD14
(sCD14); rsCD14 (2 µg/ml) was added to sCD14-depleted
plasma (sCD14 + rsCD14), or isolated plasma lipoproteins
were added to SFM (LP). The release of cell-associated
[3H]LPS was measured and expressed as a percentage of LPS
released by control plasma (mean ± S.D., number of experiments
(n) = 5, 3, 8, 8, and 3, respectively). The percentage
of cell-associated [3H]LPS released into control plasma
was 52 ± 8% (n = 13).
View larger version (20K):
[in a new window]
Fig. 5.
HDL promotes LPS release from monocyte
surfaces. A and B, PBMC were loaded with
[3H]LPS (total cell-associated [3H]LPS per
tube was 520 dpm) and incubated with R-HDL (0.1 mg of apoA-I/ml) with
or without rLBP (0.03 µg/ml) for the indicated times. After washing,
the cells were treated with cold protease solution as described under
"Experimental Procedures." A shows the percentage of the
total cell-associated [3H]LPS that could be released by
proteolysis of cell-surface proteins, and B shows the
protease-resistant fraction. Percentages were calculated from the
original cell-associated [3H]LPS. C,
CD14-transfected THP-1 cells were loaded with BODIPY-LPS as described
under "Experimental Procedures." Surface and internalized fractions
of cell-associated LPS were determined by measuring internal LPS
(presence of trypan blue) and total LPS (absence of trypan blue) by
flow cytometry before (0-4 °C) or after reincubating (30 min at
37 °C) the washed cells in rLBP (0.03 µg/ml) and rsCD14 (2 µg/ml). Surface LPS was calculated (total minus internal). The
experiments were repeated with similar results. Symbols and
error bars show mean ± range of duplicate
determinations.
, IL-1, and IL-6). As shown in Table
II, in both freshly isolated PBMC and
THP-1 cells, R-HDL reduced IL-8 release to approximately 45% of
control levels. nHDL was less inhibitory than R-HDL in this assay, in
keeping with the ability of R-HDL to promote more rapid LPS release,
yet nHDL was an effective inhibitor of cytokine responses that occurred
over a longer time period. nHDL reduced TNF-, IL-1, and IL-6
release by LPS-loaded PBMC to 49-61% of control levels; this
inhibitory effect appeared to be LPS-specific because the cellular
response to recombinant IL-1B (rIL-1) was not inhibited. Similar
degrees of response inhibition occurred after cells had been exposed to
various stimulatory concentrations of LPS. Because the measurements of
LPS release described above were performed using higher LPS
concentrations, we asked whether the rate of LPS release was different
when lower amounts of LPS had bound to the cells. We found that after
exposure to only 3 ng/ml [3H]LPS, the percentage of
cell-associated LPS released in the presence of nHDL and sCD14 in
1 h was only slightly greater than the percentage of LPS released
by cells that had been exposed to 50 ng/ml [3H]LPS (data
not shown). Although the specific activity of our [3H]LPS
precluded cell binding and release measurements at lower concentrations, we believe that the measurements of
[3H]LPS movement presented in Figs. 1-5 can be used to
describe the LPS release that occurs at low levels of cell-bound LPS
(Table II).
HDL reduces cytokine responses in LPS-loaded cells
, IL-1, and IL-6 response assays,
PBMC were pulsed with LPS (0.3-3 ng/ml), washed, and reincubated in
medium with or without nHDL (1 mg of apoA-I/ml) in the absence of LPS
transfer proteins for 16 h as described under "Experimental
Procedures." In control experiments, the cells were incubated with
rIL-1 (5-10 ng/ml) in the absence of LPS to stimulate IL-6
production. Cytokines were measured in the culture supernatants by
ELISA. The data are shown as percentage of control values (100%)
obtained from the same LPS-loaded cells that were incubated in the
absence of HDL. Means ± S.D. are shown for n
experiments done in duplicate or triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-1, IL-6, and IL-8). Inhibition of LPS
responses occurred when cell responses to a non-LPS agonist, IL-1,
were not affected. The findings thus suggest that removing LPS from the
cell surface in the presence of HDL attenuates cellular responses.
,
IL-1, and tissue factor) in whole blood requires continuous exposure
to LPS (38), and in vivo therapeutic intervention with
Limulus anti-LPS factor (39) or CD14 antibodies (40) given after LPS
challenge can rescue animals from the lethal effects of LPS. Doran
et al. (41) also reported that the addition of R-HDL
particles to whole blood attenuated LPS-induced TNF- production even
when the HDL was added up to 1 h after the LPS. Although Gallay
et al. (42) showed that brief exposure (5-10 min) of monocytes to LPS at concentrations of 10-100 ng/ml followed by incubation in 10% autologous plasma was sufficient to stimulate maximal responses, they found that lower LPS concentrations (1 ng/ml)
required longer exposure (e.g. 1 h). When cells are
exposed to very high concentrations of LPS, lipoproteins may not be
able remove a sufficiently large fraction of the LPS to decrease cell responses. The LPS concentrations used in our experiments were 3
ng/ml (Table II), and both R-HDL and nHDL reduced LPS-induced cytokine production.
View larger version (25K):
[in a new window]
Fig. 6.
LPS traffic in blood. LPS is released
from bacterial membranes and binds to leukocytes (principally monocytes
and neutrophils that express CD14) and activates them (1),
or binds to plasma lipoprotein particles (principally HDL), which
sequester (inactivate) the LPS and clear it from the circulation
(2). Lipoproteins promote the release of LPS from leukocyte
surfaces and attenuate signal responses (3).
Our findings raise the possibility that plasma lipoproteins may also be
important acceptors for other host cell-associated bacterial lipids.
For example, a recent study showed that plasma lipoproteins can bind
and neutralize lipoteichoic acids, which are thought to be important
stimulatory agonists in Gram-positive bacteria (55). Recent findings
also raise the possibility that LPS and other microbial agonists may
contribute to the inflammatory reaction in the vessel wall that occurs
during atherosclerosis (56, 57). In particular, both LPS and
LPS-containing microbes (such as Chlamydia pneumoniae) can
induce macrophages to form foam cells (58-60). In addition to the
known antioxidant properties of HDL (61, 62) and its ability to promote
cholesterol efflux from cells, our findings raise the possibility that
its antiatherogenic potency may also be related to its ability to
accept LPS (and possibly other CD14-binding ligands) from macrophages.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Peter Tobias, Rolf Thieringer, and Samuel Wright for providing recombinant LPS transfer proteins; Dr. David Spady and Tom Van Denter for help with lipoprotein chromatography; and Drs. Helen Hobbs and Jonathan Cohen for their helpful comments and suggestions.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants AI18188 and HL30086.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9113. Tel.: 214-648-6479; Fax: 214-648-9478; E-mail: rkitch@mednet.swmed.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LPS, lipopolysaccharide; HDL, high density lipoprotein; nHDL, native HDL; R-HDL, reconstituted HDL; LDL, low density lipoprotein; VLDL, very LDL; LBP, LPS-binding protein; rLBP, recombinant human LBP; rLBP-His, histidine-tagged rLBP; sCD14, soluble CD14; rsCD14, recombinant human sCD14; mCD14, membrane-bound CD14; PBMC, peripheral blood mononuclear cells; SFM, serum-free medium; BODIPY, boron dipyrromethene difluoride; apo, apolipoprotein; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin; PLTP, phospholipid transfer protein; rPLTP, recombinant PLTP; mAb, monoclonal antibody.
| |
REFERENCES |
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RESULTS
DISCUSSION
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 E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
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 T. WERNER, S. FESSELE, H. MAIER, and P. J. NELSON Computer modeling of promoter organization as a tool to study transcriptional coregulation FASEB J, July 1, 2003; 17(10): 1228 - 1237. [Abstract] [Full Text] [PDF]
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T. G. Vishnyakova, A. V. Bocharov, I. N. Baranova, Z. Chen, A. T. Remaley, G. Csako, T. L. Eggerman, and A. P. Patterson Binding and Internalization of Lipopolysaccharide by Cla-1, a Human Orthologue of Rodent Scavenger Receptor B1 J. Biol. Chem., June 13, 2003; 278(25): 22771 - 22780. [Abstract] [Full Text] [PDF]
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R. L. Kitchens and P. A. Thompson Impact of sepsis-induced changes in plasma on LPS interactions with monocytes and plasma lipoproteins: roles of soluble CD14, LBP, and acute phase lipoproteins Innate Immunity, April 1, 2003; 9(2): 113 - 118. [Abstract] [PDF]
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 J. M. Fernandez-Real, M. Broch, C. Richart, J. Vendrell, A. Lopez-Bermejo, and W. Ricart CD14 Monocyte Receptor, Involved in the Inflammatory Cascade, and Insulin Sensitivity J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1780 - 1784. [Abstract] [Full Text] [PDF]
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 R. D. Goldfarb, T. S. Parker, D. M. Levine, D. Glock, I. Akhter, A. Alkhudari, R. J. McCarthy, E. M. David, B. R. Gordon, S. D. Saal, et al. Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2003; 284(2): R550 - R557. [Abstract] [Full Text] [PDF]
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J.-M. Heinrich, M. Bernheiden, G. Minigo, K. K. Yang, C. Schutt, D. N. Mannel, and R. S. Jack The Essential Role of Lipopolysaccharide-Binding Protein in Protection of Mice Against a Peritoneal Salmonella Infection Involves the Rapid Induction of an Inflammatory Response J. Immunol., August 1, 2001; 167(3): 1624 - 1628. [Abstract] [Full Text] [PDF]
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J. H. M. Levels, P. R. Abraham, A. van den Ende, and S. J. H. van Deventer Distribution and Kinetics of Lipoprotein-Bound Endotoxin Infect. Immun., May 1, 2001; 69(5): 2821 - 2828. [Abstract] [Full Text] [PDF]
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H. W. Harris, J. E. Gosnell, and Z. L. Kumwenda Review: The lipemia of sepsis: triglyceride-rich lipoproteins as agents of innate immunity Innate Immunity, December 1, 2000; 6(6): 421 - 430. [Abstract] [PDF]
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R. L. Kitchens, P. A. Thompson, G. E. O'Keefe, and R. S. Munford Plasma constituents regulate LPS binding to, and release from, the monocyte cell surface Innate Immunity, December 1, 2000; 6(6): 477 - 482. [Abstract] [PDF]
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C. J. Vesy, R. L. Kitchens, G. Wolfbauer, J. J. Albers, and R. S. Munford Lipopolysaccharide-Binding Protein and Phospholipid Transfer Protein Release Lipopolysaccharides from Gram-Negative Bacterial Membranes Infect. Immun., May 1, 2000; 68(5): 2410 - 2417. [Abstract] [Full Text] [PDF]
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S. Viriyakosol, P. S. Tobias, R. L. Kitchens, and T. N. Kirkland MD-2 Binds to Bacterial Lipopolysaccharide J. Biol. Chem., October 5, 2001; 276(41): 38044 - 38051. [Abstract] [Full Text] [PDF]
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