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Lipid Biochemistry, Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In studies on the transport of phospholipids to HDL, Yu et al. (7) have shown that phospholipids, such as phosphatidylinositol (PI) and phosphatidylethanolamine (PE), bind to sCD14. The phospholipids bound to sCD14 can in turn be transferred to HDL. It is thus clear that CD14 shuttles not only LPS but also phospholipids.
Recent studies by Wang and Munford have confirmed the ability of CD14 to shuttle PI into cells and demonstrated that the PI becomes a substrate for phospholipase activity (8). These findings suggest a role for CD14 in the transport of phospholipids into cells.
Here we have sought evidence that CD14 may transfer phospholipids in the reverse direction, out of cells and into plasma. We show that sCD14 binds not only PI and PE but also phosphatidylserine (PS) and phosphatidylcholine (PC). We further show that cells export endogenously synthesized phospholipids to sCD14 and that mCD14 may play a role in that export. Finally, we identify the phospholipid species exported by cells to sCD14.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,2-Dioleoyl-L-3-phosphatidyl-L-[3-14C]serine,
[14C]PS (54.0 mCi/mmol) and glycerol
tri[9,10(N)- 3H]oleate,
[3H]triolein (21.0 Ci/mmol) were purchased from
Amersham Pharmacia Biotech (Piscataway, NJ).
L-
-[Myo-inositol-2-[3H](N)]phosphatidylinositol,
[3H]PI (6.61 Ci/mmol),
L--dipalmitoyl-[2-palmitoyl-9,10-[3H](N)]phosphatidylcholine,
[3H]PC (92.3 Ci/mmol),
L--1-stearoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylinositol,
[14C]PI (48 mCi/mmol),
L--1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylethanolamine,
[14C]PE (54.6 mCi/mmol), [1, 2, 6,
7(N)-3H]cholesterol,
[3H]cholesterol (84.0 Ci/mmol),
[cholesteryl-1,2,6,7(N)-3H]cholesteryl
oleate, [3H]cholesteryl oleate (84.0 Ci/mmol),
[9,10(N)-3H]palmitic acid,
[3H]palmitate (43.0 Ci/mmol),
[9,10(N)-3H]oleic acid,
[3H]oleate (5.00 Ci/mmol), and
[methyl-3H]choline chloride,
[3H]choline chloride (75.0 Ci/mmol) were
purchased from NEN Life Science Products (Boston, MA). Recombinant
human sCD141–356,
sCD141–348-His6,
sCD141–356
57–64
(sCD141–356 lacking amino acid residues from 57
to 64), and the human astrocytoma cell line, U373 MG, stably
transfected with recombinant human CD14 (U373-CD14) or with vector
(U373-neo) were gifts from Dr. Rolf Thieringer (Merck Research
Laboratories, Rahway, NJ). Because preliminary data showed that
sCD141–356 and
sCD141–348-His6 have
equivalent effects on lipid efflux,
sCD141–348-His6 was used
in most of the experiments shown here. Only Figs. 1
and 6 used
sCD141–348. Purified, lipid-free human
apolipoprotein A-I (apo A-I) was prepared as previously described
(5). Human serum albumin (HSA) was purchased from Baxter
Healthcare Corporation (Glendale, CA). The Chinese hamster ovary (CHO)
fibroblast cells, CHO-K1, stably transfected with recombinant human
CD14 (CHO-CD14) or with vector (CHO-neo) were gifts from Dr. Douglas T.
Golenbock (Boston University, MA). Agarose conjugated-monoclonal
anti-CD14, UCH-M1, was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). PMA and other reagents were purchased from Sigma (St.
Louis, MO).
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Two hundred fifty picomoles of [3H]PI or [3H]PC, or 1250 pmol of [14C]PI, [14C]PS, or [14C]PE, were dried under a stream of nitrogen, resuspended in PD-EDTA (Dulbecco’s PBS lacking Ca2+ and Mg2+ but containing 0.01% NaN3 and 1 mM EDTA), and sonicated. To adjust the specific activity to equivalence with [3H]PI, [3H]PC was mixed 1:14 (w/w) ratio with unlabeled PC from egg yolk (Sigma). The suspensions were incubated with or without 50 pmol (for [3H]-labeled phospholipids) or 250 pmol (for [14C]-labeled phospholipids) of sCD14 at 37°C for 20 h. Just before electrophoresis, an equal volume of native sample buffer (Novex, San Diego, CA) was added. Next, 661 nCi of [3H]- or 24 nCi of [14C]-labeled lipids were electrophoresed on an 8–16% gradient nondenaturing Tris-glycine polyacrylamide gel (Novex). The gel was soaked in Enlightning (NEN), dried, and exposed to XAR film (Eastman Kodak, Rochester, NY) for 32 or 48 days (bands were visible within 5 days).
Cell culture
The THP-1 human monocytic cell line was maintained in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (BioWhittaker), 100 U/ml penicillin (Sigma), and 100 µg/ml streptomycin (Sigma). Cells were kept at 37°C in a humidified atmosphere of 95% air/5% CO2. For experiments, cells were plated at a density of 2 x 105/cm2 in growth medium with 50 ng/ml PMA and incubated for 3 days to become fully differentiated macrophages.
U373-CD14 and U373-neo were grown as monolayers in RPMI 1640 with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded at a density of 3 x 104/cm2 in the growth medium and were grown overnight. CHO-CD14 and CHO-neo were grown in the same condition except that Ham’s F-12 (BioWhittaker) was used instead of RPMI 1640 and that the cells were seeded at a density of 3 x 104/cm2.
Measurement of lipid efflux from cells
Monolayers of cells grown in 48-well plates were incubated with
25 µCi/ml [3H]palmitate, 25 µCi/ml
[3H]oleate, or 5 µCi/ml
[3H]choline chloride in growth medium for
24 h (ethanol concentrations in medium did not exceed 0.5%).
After the labeling, cells were washed twice by incubation for 30 min
with RPMI 1640 (for THP-1 and U373) or Ham’s F-12 (for CHO) containing
0.1% fatty-acid free BSA, 100 U/ml penicillin, and 100 µg/ml
streptomycin, then washed three times with RPMI 1640 or Ham’s F-12.
Radioactivity of labeled lipids from THP-1 cells was normally
5,000,000 dpm per well for [3H]palmitate and
[3H]oleate labeling and 300,000 dpm per well
for [3H]choline labeling. Under these
conditions, [3H]palmitate and
[3H]oleate labeled all phospholipid classes in
the ratios expected on the basis of their known cellular abundance.
HPLC verification of this is shown in Fig. 3a for
[3H]palmitate and is not shown for
[3H]oleate.
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Analysis of lipids bound to sCD14
THP-1 cells differentiated by PMA or U373-CD14 cells were
labeled with 250 µCi/ml of [3H]palmitate in
six-well plates for 24 h. After washing as above, cells were
incubated with 10 µg/ml of sCD14 at 37°C for 30 min. The medium was
harvested and centrifuged at 14,000 x g for 5 min.
Then 1 ml of supernatant was incubated with 150 µg anti-CD14
(UCH-M1) conjugated to 75 µl agarose gel at 4°C for 16 h. The
beads were washed five time with ice-cold PBS, then the lipids bound to
sCD14 were extracted by the method of Bligh and Dyer (9).
The extracted lipids were dried under a stream of nitrogen and
resuspended in chloroform:methanol in a 2:1 solution. The lipids were
applied to an HPLC column (Kromasil 100 Silica; Higgins Analytical,
Mountain View, CA) and eluted by the method of Bünger and Pison
with slight modifications (10). Briefly, the mobile phase
reservoirs contained an A solvent of 80% dichloromethane, 19.85%
methanol, 0.15% ammonium hydroxide and a B solvent of 80% methanol,
19.85% water, 0.15% ammonium hydroxide. The following gradient was
used at a flow rate of 1.5 ml/min: 100% A from 0 to 2 min, gradient
from 0 to 20% B from 2 to 20 min, gradient from 20 to 25% B from 20
to 25 min, gradient from 25 to 40% B from 25 to 28 min, hold at 40% B
from 28 to 40 min. Radioactivity in the eluent was continuously
detected with an in-line instrument (
-RAM; IN/US Systems, Tampa,
FL). The elution positions of various phospholipid types were
determined in separate runs using radiolabeled standards.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test for the binding of a variety of lipids to sCD14, we used
radiolabeled PI, PC, PS, PE, triolein, cholesterol, and cholesteryl
oleate. The lipids were first incubated with sCD14, and lipids bound to
sCD14 were separated from unbound lipid by using native PAGE. Lipids
bound to sCD14 migrated into the gel and resolved at a lower m.w. than
unbound micellar lipids. PI, PC, PS, and PE clearly bound to sCD14
(Fig. 1
a). PI gave the highest
signal intensity among these phospholipids. Increasing the amount of
sCD14 incubated with PC yielded a dose-dependent increase of signal at
the position of sCD14 (Fig. 1b). These data confirm the
binding of PI and PS to CD14 shown in previous studies (7, 11) and offer the first direct evidence of binding of PE and PC
to sCD14. In contrast, binding of triolein, cholesterol, or cholesteryl
oleate to sCD14 was not detectable under the same conditions (data not
shown). It should be noted that binding was measured after a 20-h
incubation designed to eliminate the need for lipid transfer proteins
such as LPS binding protein.
sCD14 mediates lipid efflux from cells
We next studied the effect of sCD14 on lipid efflux from
[3H]fatty acid-labeled, PMA-differentiated
THP-1 cells. As described in Materials and Methods, the
radioactivity in these cells is distributed across the several
phospholipid classes. Labeled cells were incubated with or without
sCD14, then [3H]-labeled lipids released in the
medium were extracted and counted. The results for the cells labeled
with both [3H]palmitate and
[3H]oleate showed that 200 dpm/µg cell
protein of phospholipid was spontaneously released into the medium in
30 min (Fig. 2a). This
corresponds to 0.11% of total cellular lipids. Addition of sCD14 to
the medium dramatically increased efflux in a dose-dependent fashion to
0.62% of total lipids (Fig. 2a). The effect of sCD14 at
physiological concentrations (5 µg/ml) was statistically very
significant (p < 0.01). The efflux of lipid
labeled with either [3H]palmitate and
[3H]oleate appeared to reach completion in
30–60 min (Fig. 2, b and c). To confirm the
ability of sCD14 to promote lipid efflux, these studies were repeated
using U373-CD14 cells (data not shown). A comparable amount of efflux
and a comparable dose dependence for sCD14 were observed.
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The amount of cellular lipid released by sCD14 was calculated based on counts released and a specific radioactivity number derived from the measurement of lipid phosphorus (data not shown). These measurements indicated that, under our conditions, sCD14 in the medium was present in a >2-fold stoichiometric excess over lipid released in the medium, suggesting that the sCD14 could serve as the binding site for released lipid. To confirm that the released lipid was bound to sCD14, released lipids from [3H]palmitate-labeled THP-1 and U373-CD14 cells were subjected to immunoprecipitation with anti-CD14. Cells were labeled and incubated with sCD14 as described in Materials and Methods. Under these conditions, sCD14-dependent efflux from THP-1 and U373-CD14 cells into the medium was 305,992 ± 7,149 dpm/ml and 315,742 ± 13,463 dpm/ml, respectively. sCD14 and radioactive lipids were precipitated by this procedure, and the specificity of the precipitation was demonstrated by the finding that significant radioactivity could not be precipitated if either sCD14 was omitted from the medium or HSA was added into the medium instead of sCD14 (data not shown). Approximately 10% of the total radioactivity specifically exported by sCD14 into the medium was precipitated with anti-CD14. We attribute this relatively low proportion of radiolabeled lipid to losses during immunoprecipitation. Lipid could be lost through incomplete precipitation of the sCD14 and from disassociation of phospholipids from sCD14 during washing of the agarose.
HPLC analysis of the labeled lipids in cell extracts of THP-1 and
U373-CD14 showed the expected constituents of lipids with a dominant
constituent of PC (Fig. 3, a
and c). Lipids precipitated with anti-CD14 were
extracted in parallel and subjected to HPLC to determine the lipid
species present (Fig. 3, b and d). These
experiments showed that PC was also the dominant lipid species bound by
sCD14. This finding is consistent with the binding of PC to sCD14 shown
in Fig. 1. In addition to PC, small amounts of other lipid species also
precipitated with sCD14. These roughly mirror the bulk lipid
composition of the source cells. One exception is the presence of free
palmitate in the materials immunoprecipitated by anti-CD14 but not
in the source THP-1 cells. This might arise from hydrolysis during
immunoprecipitation and could contribute to the relatively low
efficiency of precipitation of lipid.
Efflux of PC to sCD14
To confirm the ability of sCD14 to promote export of PC, we
labeled the phospholipids by incubating cells with
[3H]choline chloride. At the same time, we
compared the efficiency of phospholipid efflux by sCD14 with that of a
well-characterized acceptor of phospholipids, apo A-I, and with a
control protein, HSA. Labeled THP-1 cells were washed and incubated
with or without sCD14, apo A-I ,or HSA for 30 min. Labeled lipids
exported into the medium were extracted and counted. As with cells
labeled with palmitate or oleate, sCD14 could facilitate efflux of
[3H]choline-labeled lipids in a dose-dependent
manner (Fig. 4), thus confirming the
ability of sCD14 to promote efflux of PC. In contrast, the control
protein HSA had very little effect on
[3H]choline-labeled lipid efflux even at the
high concentration of 50 µg/ml. The amount of
[3H]choline-labeled lipid efflux to sCD14 and
to apo A-I was comparable at 15 µg/ml, while efflux to apo A-I
clearly exceeded that to sCD14 when the proteins were used at 50
µg/ml. The ability of sCD14 to mediate lipid efflux on a scale
comparable with apo A-I suggests an important role of CD14 in cellular
phospholipid efflux.
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We have previously demonstrated that LPS can very rapidly exchange
between CD14 molecules (12) and that mCD14 may accelerate
LPS uptake from sCD14 (4, 13). Because PMA-differentiated
THP-1 cells express mCD14 (14), we asked whether mCD14
played a critical role in lipid efflux to sCD14. To do so, we used
clones from two types of cell lines, U373 and CHO. We used control
U373-neo and CHO-neo clones that do not express mCD14 and the
mCD14-expressing clones U373-CD14 and CHO-CD14. The lipid efflux
measured from cells labeled with [3H]palmitate,
[3H]oleate, or
[3H]choline chloride is shown in Fig. 5. With U373 cells, sCD14-dependent lipid
efflux was higher on mCD14 expressing cells than on control cells (Fig. 5, a–c). This was most evident in studies using
[3H]choline-labeled cells (Fig. 5c).
These data suggest that mCD14 may contribute to phospholipid efflux.
However, in CHO cells, significant lipid efflux was observed in CHO-neo
cells, and expression of mCD14 appeared to reduce the efflux of lipids
mediated by sCD14 (Fig. 5, d–f). These data suggest that
the role of mCD14 in lipid efflux may depend critically on the
cell type.
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Amino acid residues from 57 to 64 of sCD14 have been shown to be
part of an LPS binding site (15). To ask whether the LPS
binding site is necessary for phospholipid efflux to sCD14, we employed
the mutant sCD1457–64,
which bears a deletion of the critical residues. Significantly less
efflux of phospholipid to
sCD1457–64 was observed
using [3H]choline,
[3H]palmitate, or
[3H]oleate labeling, although efflux was not
completely blocked (Fig. 6). These data
suggest at least a partial contribution of the LPS binding site to PC
efflux.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); 2)
anti-CD14 precipitated both CD14 and PC from the supernatants of
cells with radiolabeled phospholipids (Fig. 3); and 3) sCD14 caused
efflux of choline-labeled phospholipids from cells (Fig. 4). These
studies suggest a role for sCD14 in transport of the dominant lipid
component of cell membranes and lipoproteins, PC.
Additional studies indicated that PC in the membranes of cells may pass
readily to a binding site on sCD14 in the medium. The amount of lipid
that passes out of cells in this way rises linearly with the
concentration of sCD14 in the medium (Fig. 1a), suggesting
that sCD14 availability, not cellular processes, are limiting for this
event. Moreover, the efflux is rapid, reaching equilibrium by 30 min
(Fig. 1, b and c). These data suggest that CD14
may provide a conduit for lipids out of cells and into the plasma. A
role of sCD14 in transport of common lipids is further suggested by its
abundance. With a plasma concentration of 3 µg/ml, sCD14 is in
great molar excess (1000-fold) over the plasma concentrations of LPS
even during acute sepsis (1).
Previous work from Wang and Munford has documented a role for CD14 in the transport of PI from extracellular depots into cells (8). Therefore, it is clear that CD14 can mediate bidirectional flux of phospholipids, both into and out of cell membranes. We may therefore ask which of these processes, influx or efflux, predominates in vivo. Several observations suggest that efflux may be the most traveled route. Plasma contains lipoproteins, such as HDL, which transport phospholipids and may serve as acceptors of cellular phospholipids. Previous work from our group has demonstrated that sCD14 readily donates both LPS and phospholipids to HDL particles (5, 7). sCD14 may thus shuttle phospholipids from peripheral cells to HDL particles for eventual transport to the liver. This route of lipid transport to the liver is suggested by a large body of work on cholesterol, which is known to follow this precise path: cells cannot degrade cholesterol, and all excess cholesterol synthesized is removed from peripheral cells and brought to the liver by a process known as "reverse cholesterol transport." Additional data also argue for net movement of phospholipids to HDL. Plasma contains several enzymes, including hepatic lipase, soluble phospholipase A2, and lecithin-cholesterol acyl transferase, which act to destroy PC in HDL. Therefore, net movement of PC to HDL is needed to account for the destruction of PC by these enzymes.
Our studies suggest two routes for transfer of PC from cells to sCD14,
one that is direct and one that involves mCD14. In U373 cells, efflux
of lipid to sCD14 is strongly enhanced by expression of mCD14 (Fig. 5).
The ability of mCD14 to enhance efflux of phospholipids to sCD14
appears analogous to the ability of mCD14 to enhance influx of LPS from
sCD14 to cells that has been previously documented (4, 13). In both cases, we postulate that the lipids bind to mCD14
en route to or from sCD14. Previous work from our laboratory indicates
that LPS exchanges between CD14 molecules very rapidly and without the
requirement for a lipid transfer protein (12). Therefore,
we would expect that the rate-limiting step in efflux from U373 cells
is transport of phospholipids from the plasma membrane to mCD14.
Previous work has suggested the existence of a lipid transporter with
such an activity. Vasselon et al. have shown that monocytes express a
protease-sensitive activity necessary for mCD14-dependent influx of LPS
(13). It thus appears likely that U373 cells use a
mCD14-dependent pathway of lipid efflux because they possess a
transporter that rapidly transfers phospholipids to mCD14 in preference
to sCD14.
In contrast with the findings on U373 cells, we observed that phospholipid efflux was not enhanced by mCD14 expression on CHO cells. We conclude that phospholipids may exit CHO cells directly to sCD14, without mCD14 as an intermediate. In keeping with the argument above, we may speculate that CHO cells do not express a lipid transfer protein specific for transport to and from mCD14. Rather, they may have a transporter that can directly transfer phospholipids to and from sCD14.
PC effluxed from cells to sCD14 is likely to be in equilibrium with the PC in HDL particles. The role of sCD14 in transport of lipids to HDL is the topic of ongoing studies.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, and Division of Bacterial Toxin, Research Center for Infectious Disease, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
3 Address correspondence and reprint requests to Dr. Samuel D. Wright, Merck Research Laboratories, 126 East Lincoln Avenue, R80W-250, Rahway, NJ 07065.
4 Abbreviations used in this paper: mCD14, membrane-bound CD14; sCD14, soluble CD14; HDL, high density lipoprotein; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; apo A-I, apolipoprotein A-I; HSA, human serum albumin; native PAGE, nondenaturing PAGE; PD-EDTA, Dulbecco’s PBS lacking Ca2+ and Mg2+ but containing 0.01% NaN3 and 1 mM EDTA; CHO, Chinese hamster ovary.
Received for publication July 20, 2000. Accepted for publication October 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)- or
[3H]oleate (
)-labeled differentiated THP-1 cells were
incubated with the indicated concentration of sCD14 in RPMI 1640 for 30
min. Each point represents the mean of three measurements ± SD.
b and c, [3H]Palmitate
(b)- or [3H]oleate
(c)-labeled THP-1 cells were incubated with (•) or
without (
) 10 µg/ml sCD14 in RPMI 1640. Each point represents the
mean of three measurements ± SD. The increase in efflux in the
presence of sCD14 was considered sCD14-dependent efflux (
).
), or HSA (