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J. Biol. Chem., Vol. 277, Issue 10, 7970-7978, March 8, 2002
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From the
Received for publication, October 4, 2001, and in revised form, December 26, 2001
The bactericidal/permeability increasing (BPI)
and lipopolysaccharide (LPS)-binding (LBP) proteins are
closely related two-domain proteins in which LPS binding is mediated by
the NH2-terminal domain. To further define the role
of the COOH-terminal domain of these proteins in delivery of LPS to
specific host acceptors, we have compared interactions of LBP, BPI,
LBPN-BPIC (NH2-terminal domain of
LBP, COOH-terminal domain of BPI), and
BPIN-LBPC with purified 3H-LPS and,
subsequently, with purified leukocytes and soluble (s)CD14. The
COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both
polymorphonuclear leukocytes and monocytes resulting in cell
activation. In the presence of Ca2+ and Mg2+,
LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent Mr > 20 × 106 Da), but only LPS associated with LBP and
BPIN-LBPC is disaggregated in the presence of
CD14. BPI and LBPN-BPIC promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these
closely related endotoxin-binding proteins dictates the route and host
responses to complexes they form with endotoxin.
Bacterial invasion triggers sensitive, specific molecular alarms
that result in the mobilization of host defenses designed to recognize
and eliminate invading bacteria and their remnants. The major inducer
of host responses to Gram-negative bacteria (GNB)1 is endotoxin,
i.e. lipopolysaccharide, LPS, the unique glycolipid that
comprises the bulk of the outer leaflet of the GNB outer membrane (1).
These amphipathic molecules are composed of a unique, conserved
hydrophobic moiety, the lipid A region, that is a disaccharide of
N-acetylglucosamine substituted with saturated fatty acids
and attached to a highly charged acidic carbohydrate region of varying
size and composition (1, 2). The chemical structure of endotoxin
promotes the formation of highly ordered, but potentially malleable
aggregated state(s). Interaction with host endotoxin-binding proteins
alters the physical presentation of endotoxin and its ability to
activate cell responses (2-5). The cumulative work of many
laboratories has implicated lipopolysaccharide-binding protein (LBP),
CD14, Toll-like receptor 4, and MD-2 as key factors in cell
activation by LPS (3, 6-13). LBP facilitates delivery of LPS to both
membrane-bound, GPI-linked (mCD14) and soluble CD14 (sCD14)
(14-19). Through interaction with CD14, LPS activates cells via a
transmembrane receptor capable of promoting signal transduction. This
recognition/response cascade includes the Toll-like receptor family of
proteins, most notably Toll-like receptor 4, that serve as the primary
mediator of endotoxin signaling (20, 21).
The host also utilizes defense mechanisms that blunt
endotoxin-triggered inflammatory responses by eliminating viable GNB and by neutralizing endotoxin. One potent host protein that blocks LPS
activity is bactericidal/permeability increasing protein (BPI), a basic
protein residing in azurophilic granules of polymorphonuclear (PMN)
leukocytes and in the extracellular fluid of PMN-rich inflammatory exudates (22). BPI efficiently neutralizes LPS and is also potently cytotoxic and opsonic, especially toward GNB (22-25). Although the
functional properties of LBP and BPI differ markedly, they share 45%
amino acid identity, are encoded within the same region of chromosome
20, and belong to a family of lipid-binding proteins that include
phospholipid transfer protein and cholesteryl ester transfer protein
(26-30). The three-dimensional crystal structure for BPI reveals an
unusual "hinged" two-domain boomerang-like molecular structure. The
extensive sequence homology between LBP and BPI predicts a nearly
superimposable three-dimensional structure for LBP (30) suggesting that
despite their different bioactivities, BPI and LBP have a similar
organization of structure and function.
In support of this view, studies on the interaction of LBP and BPI with
LPS have demonstrated that the NH2-terminal domain of each
protein is responsible for binding LPS (28, 31-34). In BPI, both
affinity for LPS and antibacterial activity are concentrated in this
portion of the protein (31-33, 35, 36). In contrast, promotion by LBP
of CD14-dependent cell activation by LPS requires the
complete LBP molecule implying that after association of LBP with LPS,
the COOH-terminal domain of LBP is needed for transfer of LPS to CD14
(37, 38). Similarly, the ability of BPI to promote delivery of intact
GNB to PMN depends upon both the NH2- and COOH-terminal
domains of BPI (23). Thus, protein binding to aggregated endotoxin is
mediated by the NH2-terminal domain of LBP and BPI,
followed by delivery of LPS to distinct host acceptors via their
COOH-terminal domains. We speculate that the functional properties of
endotoxin-containing particles associated with BPI or LBP could be
determined, in part, by differences in the identity and/or cellular
localization of their downstream targets.
In this study, LBP, BPI, and previously described chimeric proteins of
LBP and BPI (39) have been utilized with metabolically labeled
preparations of Escherichia coli K12 LPS (40) to further define the role of the COOH-terminal domain of BPI and LBP in delivery
of endotoxin to specific cellular and extracellular host targets. We
compared the effects of these proteins on the binding of LPS to and
activation of enriched populations of leukocytes and on the interaction
of LPS with sCD14. A gel filtration chromatography system has been
applied that permits isolation of a homogeneous population of LPS
aggregates (LPSagg). Using this gel filtration system, we
monitored changes in the physical state of LPSagg with LBP,
BPI, or chimeric proteins and sCD14. Our results indicate that both the
targeting of LPS to specific host acceptors and the subsequent
functional effects depend mainly on the properties of the
carboxyl-terminal domain of BPI and LBP.
Materials--
The following proteins were provided by Dr.
Stephen Carroll, Xoma Corp. (Berkeley, CA): recombinant human BPI
(rBPI), human BPI-21, the bioactive NH2-terminal fragment
of BPI (residues 1-193), LBP, sCD14, and two chimeric proteins,
LBPN-BPIC (amino acids 1-197 of LBP and
200-456 of BPI) and BPIN-LBPC (amino acids
1-200 of BPI and 199-456 of LBP), and rabbit polyclonal anti-LBP
antibodies. Isolation of native human BPI and LBP and the design and
purification of the recombinant proteins have been described previously
(39). Antibodies against BPI were generated in goat as has previously been described (41). Antibodies against CD14 utilized were MEM-18, MY4,
and FITC-MY4 from Coulter, Inc. Sephacryl HR S500 was obtained from
Amersham Biosciences, Inc. (Piscataway, NJ). Lucigenin was purchased
from Sigma. Human serum albumin (HSA) was endotoxin-free, 25% stock
solution manufactured by Baxter Healthcare Corp., Glendale, CA.
3H-LPS (purchased from List Biological Laboratories,
Campbell, CA) was dissolved in endotoxin-free water to a final
concentration of 100 µg/ml and sonicated twice on ice for 10 min.
Aliquots were stored frozen until needed or aggregates were immediately
isolated by column chromatography on Sephacryl S500 as described below. Samples for isolation of a homogeneous 3H-LPS aggregate
population were diluted 1:1 in Hanks' balanced salts buffer solution
containing magnesium and calcium (HBSS+), 20 mM HEPES with
2% HSA and incubated at 37 °C for 15 min before application to the
column and elution as described below. Essentially all (>98%)
3H cpm in purified LPSagg are present within
the fatty acids specific to the lipid A region of LPS (42).
Cell Preparation--
Venous blood was drawn from healthy human
volunteers, after informed consent in accordance with the established
guideline (IRB committee approved protocol), into a heparinized
syringe, and leukocytes were isolated by sedimentation in pyrogen-free 3% dextran (U. S. Biochemical Corp.). The platelet- and
leukocyte-rich upper phase were layered on Histopaque (Sigma) in a 2:1
ratio (v/v) and spun at 50 × g at room temperature for
30 min. Mononuclear cells were collected from the interface between the
upper serum/dextran phase and the lower Histopaque phase. The cell
pellet contained PMN and erythrocytes; the latter were subsequently
removed by hypotonic lysis. The purified PMN and mononuclear cells
(MNC) were washed in Hanks' balanced salts solution without magnesium and calcium (HBSS
To obtain a more purified monocyte cell fraction, the mononuclear cell
mixture obtained after Histopaque sedimentation was incubated with
immunomagnetic beads bearing the anti-CD3 (pan T-cell marker) and
anti-CD19 (pan B-cell marker) in 4:1 bead:lymphocyte ratio according to
the manufacturer's instructions (Dynal Corp., Lake Success, NY) for
negative selection of monocytes. After 30 min at room temperature,
lymphocytes bound to the magnetic beads were removed by application of
the magnet to the tube. The non-adherent cells were decanted into a
fresh tube and diluted in HBSS+, 1% HSA to 0.5-1 × 106 cells/ml. These cells were histologically >85%
monocytes and >75% CD14+ as evaluated by fluorescence-activated cell
sorter analysis using FITC-MY4.
Cell Binding Assays--
3H-LPS (specific activity
900 cpm/ng) from E. coli K12 LCD25 (List Biologicals) was
utilized in binding studies to human PMN or enriched MNC as has
been previously described (23). Typical incubation mixtures contained
cells (2.5-5 × 106) and 100 ng/ml 3H-LPS
in 0.5 ml HBSS+, 1% HSA ± the indicated protein in a dose range
from 0.1 to 100 nM. Incubations were for 1 h at
37 °C. After incubation, cells were washed twice in cold saline,
transferred in saline to a fresh tube, centrifuged at 500 × g for 4 min at 4 °C (42), and the pellet solubilized by
boiling in 100 µl of 5% SDS, 10 mM EDTA for 10 min.
Recovered 3H-LPS in the solubilized cells was measured by
liquid scintillation spectroscopy in a Beckman liquid scintillation
counter. Cell associated 3H-LPS is expressed as percent of
total added 3H-LPS cpm.
In experiments examining the effect of anti-CD14 antibodies, 10 µg/ml
MEM-18 or MY-4 was incubated for 30 min at room temperature with cells
before the addition of 3H-LPS and proteins (LBP, BPI,
LBPN-BPIC, and
BPIN-LBPC). Binding was determined as described above.
Sephacryl HR S500 Chromatography--
Columns of Sephacryl S500
(1.5 × 18 cm) were routinely pre-equilibrated in HBSS+, 10 mM HEPES, 1% HSA, pH 7.4. In experiments examining the effect of the divalent cations Mg2+ and
Ca2+, samples were prepared in Hanks' balanced salts
buffer minus Mg2+ and Ca2+, 10 mM
HEPES, 5 mM EDTA, 1% HSA and eluted from columns in the same buffer. Samples applied to the columns were eluted at room temperature at a flow-rate of 0.5 ml/min. Fractions (1 ml) were collected, and aliquots evaluated for 3H-LPS content by
liquid scintillation spectroscopy to determine elution profiles. A
homogeneous population of 3H-LPS aggregates was prepared by
chromatography on Sephacryl S500 HR of 20 µg of 3H-LPS
reconstituted as described above. Peak fractions (LPSagg, Mr~ 1-1.5 × 106 Da) were
combined and used as a source of 3H-LPS of a defined size
range in subsequent experiments. Samples of 3H-LPS ± indicated proteins (BPI, LBP, BPIN-LBPC,
LBPN-BPIC ± sCD14) were incubated for 60 min
at 37 °C in HBSS+, 1% HSA in volumes from 100 to 500 µl before
application to columns. The purified aggregates either were used
immediately or stored in Teflon vials. These samples were stable at
4 °C for at least 2 weeks. For binding assays of LPSagg,
LBP:LPSagg, and BPI:LPSagg, the peak fractions obtained from Sephacryl S500 chromatography were pooled, evaluated for
radioactivity, and an aliquot rerun to confirm homogeneity of pooled
fractions. Because of the low concentration of LPS in these fractions,
binding assays typically were done in a total volume of 1 ml. Molecular
size of aggregates was determined by comparing elution of the
aggregates against that of the following standard proteins run under
the same conditions +/
To verify the presence of LBP or BPI in isolated
LPSagg:protein aggregates, samples of LPSagg + LBP or BPI were incubated as described and the aggregates isolated in
the void volume of Sephacryl S500 columns equilibrated in Hanks', 10 mM HEPES (1.5 × 6 cm). Shorter columns were used to
reduce losses of LPSagg during chromatography without
albumin yet allowed efficient isolation of the large aggregates. Void
volume fractions containing LPS were precipitated with tricholoracetic
acid, the precipitated material was washed, and resuspended in SDS-PAGE
sample buffer. Control samples containing only LBP or BPI with no LPS
were also chromatographed and treated in a similar manner. In addition
to these samples, control samples of LBP and BPI of varying
concentration were electrophoresed using an Amersham Biosciences, Inc.
PhastGel System through either 12.5 (LBP) or 10-15% (BPI) acrylamide
gels and transferred to nitrocellulose by semi-dry transfer using the same system. For immunoblotting, the nitrocellulose was washed with
phosphate-buffered saline containing 0.05% Tween 20 and then blocked
with 3% bovine serum albumin in the same buffer for 1 h at
25 °C. After washing, the blots were treated with the appropriate primary antibody (1:1000 rabbit anti-LBP or goat anti-BPI serum) diluted in 1% bovine serum albumin, phosphate-buffered saline, 0.05%
Tween 20 overnight at 25 °C. After washing with phosphate-buffered saline, 0.05% Tween 20, the blots were incubated with secondary antibody conjugated to horseradish peroxidase (either donkey
anti-rabbit IgG or rabbit anti-goat IgG) for 1 h at 25 °C,
washed with phosphate-buffered saline, 0.05% Tween 20. Then the blots
were developed using the Pierce SuperSignal substrate system.
Chemiluminescence--
3H-LPS (100 ng/ml) ± LBP or BPI or purified aggregates of 3H-LPS as indicated (2 ng/ml in 90 µl) were preincubated for 30 min at 37 °C. Samples
were added to 96-well plates (Optiplate, Packard) followed by 10 µl
of a cell suspension containing 30 or 60 µM lucigenin and
either 5 × 106/ml monocytes isolated by
immunomagnetic beads or 1 × 107/ml MNC from
Histopaque sedimentation (final volume: 100 µl). Lower concentrations
of lucigenin were used in assays with 100 ng LPS/ml to reduce the
sensitivity of LPS-triggered lucigenin-enhanced chemiluminescence.
Chemiluminescence was measured at 37 °C with 5 s of gentle
shaking every 5 min in an Anthos Lucy I luminometer (Columbia
Bioscience, Federick, MD). Data were collected for 5 s/sample at 10-min
intervals for 2 h. LPS-dependent chemiluminescence, reflecting increased cellular oxidase activity, was measured using a
dose curve of unfractionated LPS (0.1-1000 ng LPS/ml) or purified LPSagg (about 0.1-20 ng/ml). Leukocyte chemiluminescence
was negligible (<100 photons/s) in the absence of LPS.
Delivery of [3H]LPS-mediated by LBP, BPI, and LBP/BPI
Chimera to PMN and Monocytes--
The ability of endotoxin-binding
proteins to facilitate interaction and delivery of purified
3H-LPS to peripheral blood leukocytes was evaluated by
measuring cell association of 3H-LPS in the presence of
varied concentrations of LBP, BPI, BPI-21, and two chimeric LBP/BPI
proteins, LBPN-BPIC and
BPIN-LBPC. Two cell populations were examined:
PMN (>98% pure) and a purified MNC that contained
Therefore, proteins containing the carboxyl-terminal domain of LBP
(LBP, BPIN-LBPC) promoted delivery of purified
LPS to both MNC and PMN (MNC > PMN). Proteins containing the
carboxyl-terminal domain of BPI (BPI,
LBPN-BPIC) only promoted LPS association with MNC. BPI-21 did not promote 3H-LPS cell association to
either PMN or MNC (Fig. 1B) indicating a requirement of the
carboxyl-terminal domain of BPI for BPI-dependent delivery
of endotoxin to MNC.
Both BPI as well as LBP-dependent 3H-LPS
binding observed in MNC are apparently to the monocytes in MNC.
3H-LPS binding was increased when a more monocyte-enriched
MNC (>85%) was used (data not shown). In contrast, there was little or no LPS binding to purified lymphocytes in the absence or presence of
LBP or BPI. Fluorescence-activated cell sorter analysis of MNC
incubated with BODIPY-endotoxin indicated that both LBP- and BPI-dependent binding of 3H-LPS to blood
mononuclear cells is restricted to
monocytes.2
Role of CD14 in LBP, BPI, and Chimeric Protein-mediated Delivery of
LPS to MNC--
On monocytes and PMN, mCD14 is the major initial
target of LPS exposed to LBP (9, 42, 43). Differences in delivery of
3H-LPS to PMN and monocytes by LBP, BPI, and the two
chimera (Fig. 1) could be explained by: 1) differences in mCD14 levels
in monocytes and PMN (monocytes > PMN) (42, 44); or 2)
involvement of different acceptor molecules for LPS associated with LBP
or BPIN-LBPC versus BPI or
LBPN-BPIC. To test the role of mCD14, we
measured the effects of two anti-CD14 monoclonal antibodies (mAb) on
cell association of LPS. The two mAbs, MY-4 and MEM-18, are directed
against different epitopes of CD14 (45, 46). They were preincubated
with the cells prior to addition of 3H-LPS and the
indicated endotoxin-binding proteins. As shown in Fig.
2A, both MY-4 and MEM-18
almost completely blocked delivery of LPS to MNC (or to PMN; data not
shown) that was mediated by LBP and BPIN-LBPC.
These same mAbs had no (MY-4) or more limited (MEM-18) effect on LPS
association mediated by BPI or LBPN-BPIC to
MNC. These findings suggest that LPS binding to MNC and PMN is
dependent on mCD14 when mediated by proteins containing the COOH-terminal domain of LBP. In contrast, LPS binding to MNC mediated by proteins containing the COOH-terminal domain of BPI is largely independent of mCD14.
The COOH-terminal Domain of LBP Is Needed for
sCD14-dependent Disaggregation of LPS--
An alternative
interpretation of the above findings is that LPS complexes associated
with proteins containing the COOH-terminal domain of BPI engage CD14
but in a way that is less susceptible to inhibition by the particular
anti-CD14 mAb examined. To further test the possible role of the
COOH-terminal domain of LBP and BPI in interactions of LPS with CD14,
we monitored these interactions in solution using sCD14 and a gel
filtration system (5) for the separation of protein-LPS complexes and
aggregates. To facilitate these analyses, a subpopulation of
3H-LPS aggregates was isolated by Sephacryl S500 HR gel
filtration of commercially prepared 3H-LPS from E. coli K12 LCD25 (Fig. 3). Gel
filtration chromatography was carried out using buffer conditions
compatible with bioassays to permit a direct juxtaposition of the
physical and functional effects of protein interactions with LPS. The
recovered subpopulation of LPSagg yielded a symmetrical
peak with an apparent size Mr ~1-1.5 × 106 upon re-chromatography (Fig. 3). These aggregates were
stable at 4 °C for at least 1 month.
Incubation of LPSagg with LBP produced aggregates of LPS
with an increased apparent size (
Treatment of LPSagg with BPI also resulted in aggregates of
LPS of increased apparent size comparable with that observed with LBP
(Fig. 4). These large aggregates contained BPI as shown by immunoblot
analysis of isolated BPI:LPSagg (Fig. 4C). In
contrast to LBP:LPSagg, BPI:LPSagg do not
disaggregate to a smaller complex during incubation with sCD14
treatment (Fig. 6). Co-incubation of LBP
or BPI and sCD14 with purified LPSagg (~1-2 × 106 Da) produced similar results, i.e. small
complexes (~1 × 105 Da) in the presence of LBP
(Fig. 5B), but larger aggregates (
The contribution of the carboxyl-terminal domain of these
endotoxin-binding proteins to CD14-promoted disaggregation of isolated LPSagg was monitored by changes in the physical state of
isolated LPSagg promoted by interaction with
BPIN-LBPC and
LBPN-BPIC. As shown in Fig.
7, BPIN-LBPC
facilitated sCD14-dependent disaggregation of
LPSagg, whereas LBPN-BPIC did not.
The results indicate that whereas the COOH-terminal domains of LBP and
BPI each contribute to LPS association with MNC, only the COOH-terminal
domain of LBP has the capacity to deliver LPS to (s)CD14 in such a
manner that results in disaggregation.
Role of the COOH-terminal Domain of LBP in sCD14-independent
Disaggregation of LPS in the Presence of EDTA--
The findings
described above demonstrate appreciable disaggregation of LPS only when
an endotoxin-binding protein containing the COOH-terminal domain of LBP
and, in addition, sCD14 are both present. These results differ from
many earlier studies that have suggested direct disaggregating effects
of LBP on LPS. In contrast to our studies, these earlier studies were
done in the absence of divalent cations (i.e.
Mg2+ and Ca2+) (8, 19, 47, 48, 50) needed to
maintain close packing of the highly anionic LPS (51-53). Therefore,
we repeated the analysis in the presence of EDTA of the effects of LBP
on the aggregation state of LPS as assessed by gel sieving. Incubation
of the purified LPSagg with EDTA alone induced
heterogeneity among the LPS aggregates but did not cause marked
disaggregation (Fig. 8A). In
contrast, under these divalent cation-free conditions, LBP produced
dramatic disaggregating effects (Fig. 8A) whereas BPI still
increased the size of LPS aggregates (Fig. 8B). The
contrasting CD14-independent effects of LBP and BPI on LPS aggregates
seen under these conditions conform closely to previous observations
made utilizing density gradient centrifugation and light scattering
(47). Proteins containing the COOH-terminal domain of LBP produced
greater disaggregation of LPS than the corresponding proteins
containing the COOH-terminal domain of BPI (i.e. LBP > LBPN-BPIC (Fig. 8A);
BPIN-LBPC > BPI (Fig. 8B)). These
findings therefore indicate a role of the COOH-terminal domain of LBP
in both sCD14-independent LPS disaggregation in the presence of EDTA as
well as sCD14-dependent LPS disaggregation in the presence
of Ca2+ and Mg2+.
Effect of LBP and BPI on Activation of Monocytes by
LPS--
Previous studies have demonstrated that the
LPS-dependent lucigenin-enhanced chemiluminescence response
of a mixed leukocyte population (comprised primarily of PMN) was
stimulated by LBP and BPIN-LBPC, but inhibited
by BPI, LBPN-BPIC, and rBPI21 (23). The data presented here that demonstrate BPI and
LBPN-BPIC dependent delivery of LPS to
monocytes, but no delivery to PMN, prompted us to re-examine the effect
of BPI on LPS-triggered cell activation using a monocyte-enriched cell
population. Under identical conditions to those utilized in the binding
assays, LBP enhanced cellular response to LPS. In contrast, BPI caused
a dose dependent decrease in lucigenin-enhanced chemiluminescence (Fig.
9). At the protein concentrations tested
here, neither LBP nor BPI in the absence of LPSagg, had any
effect on lucigenin-enhanced chemiluminescence.
A more direct assessment of the bioactivities of LBP:LPSagg
and BPI:LPSagg was carried out with the large protein-LPS
aggregates ( Although LBP and BPI are closely related by sequence and predicted
three-dimensional structure, they often display opposing effects on
host cell-endotoxin interactions (22, 23, 39, 47). Previous studies
have strongly suggested that the stimulatory effects on
CD14-dependent cell activation by LPS, produced by LBP but
not by BPI, depend on the ability of LBP to promote delivery of LPS to
and disaggregation by CD14 (5, 19, 48, 49). Comparison of the effects
of native and variant forms of BPI and LBP, including BPI/LBP chimeras,
have led to the speculation that the COOH-terminal domain of LBP plays
an essential role in this process (23, 37-39). We now provide
experimental data that support this contention. Independent of the
origin (BPI versus LBP) of the NH2-terminal
domain that initially engages LPS, delivery of LPS to cells via mCD14
(Fig. 2) and disaggregation of LPS by sCD14 (Figs. 5-7) depend upon
the presence of the COOH-terminal domain of LBP and closely correlate
with cell activation (5, 23). The 3H-LPS containing
complexes generated during incubation with LBP or
BPIN-LBPC and sCD14 elute with an apparent size
of ~1 × 105 Da during Sephacryl S500 gel filtration
chromatography. These species closely resemble potent bioactive
sCD14-endotoxin complexes formed under essentially identical
experimental conditions with other endotoxin species (5). Although it
is not yet possible to test for physical transformations of endotoxin
after interaction with mCD14, similar changes may be necessary for
maximal mCD14 as well as sCD14-dependent cell activation.
In contrast to sCD14-dependent disaggregation of LPS,
incubation of LPS aggregates (~1-1.5 × 106 Da)
with either LBP or BPI alone produced an increase in apparent aggregate
size (Fig. 4). Both LBP:LPSagg and BPI:LPSagg
are eluted in the void volume during Sephacryl S500 chromatography
suggesting a molecular size of Initial interactions of LBP and BPI with LPS are driven by
electrostatic attractions between anionic moieties of LPS concentrated near the lipid A region and basic amino acids clustered at the extreme
end of the NH2-terminal domain (22, 30, 54). In BPI, these
sites are contiguous with an extended cationic protein surface whereas
in LBP this adjacent surface is acidic (30, 55).4 These differences in
the density and distribution of protein charges probably account for
the stronger attraction of LPS for BPI than for LBP and could
contribute to the greater facility of LBP:LPSagg to extract
and release CD14-endotoxin complexes and the inability of
BPI:LPSagg to release LPS
(5).5 This hypothesis is
supported by the observed greater instability of LBP:LPSagg
versus BPI:LPSagg (Figs. 5 and 6) and greater
efficiency of LBP versus BPIN-LBPC
in sCD14-dependent (Figs. 5 and 7) and sCD14-independent
(Fig. 8) disaggregation. Therefore, differences in interactions with
LPS mediated by the NH2-terminal domain of LBP and BPI may
further contribute to the efficiency of LBP-promoted LPS disaggregation
and cell activation.
It has been generally presumed that the inhibitory effects of BPI on
LPS signaling reflect the inability of BPI-endotoxin complexes to
interact with endotoxin-responsive cells. The absence of detectable BPI
or LBPN-BPIC-dependent binding of
LPS to PMN (Fig. 1A) is consistent with that view. However,
an earlier study suggested BPI-dependent association of LPS
with promonocyte-like THP-1 cells (56, 57). We have extended that
observation to mature monocytes. The fact that
BPI-dependent LPS association is promoted by BPI or
LBPN-BPIC, but not BPI21, reveals
that the COOH-terminal domain of BPI is instrumental in
BPI-dependent delivery of LPS to these cells. Delivery of
BPI-endotoxin aggregates does not provoke the same degree of cellular
response elicited by LBP-endotoxin aggregates. Differences in
interactions with CD14 as specified by different properties of the
COOH-terminal domains of LBP and BPI (see Figs. 2 and 7) could explain
the different functional consequences of LPS delivery to monocytes via
LBP and BPI. The partial inhibitory effect of one particular mAb to
CD14, MEM18, on BPI or LBPN-BPIC delivery of
LPS to monocytes raises the possibility that BPI:LPSagg
transiently engages CD14, but in a manner insufficient to generate the
CD14-endotoxin complexes responsible for maximal cell activation. The
effects of the MEM-18 mAb could be nonspecific, for example, sterically
impeding BPI-dependent delivery of LPS to a neighboring
acceptor molecule. The versatility of CD14 in its associations with
diverse extracellular microbial and host ligands and with host plasma
membrane constituents makes either scenario a possibility (15, 58-66).
It is noteworthy that a region in CD14 essential for MEM18 binding
(amino acids 57-64) (46) may represent a flexible bridge important in
intra- and intermolecular interactions.
We have previously shown that BPI and, to a lesser extent, BPI-21,
promote uptake of encapsulated Gram-negative bacteria by neutrophils
(23).6 These data as well as
the findings reported in this study indicate a potential role of BPI in
clearance of bacteria and bacterial remnants, each dependent on the
COOH-terminal domain of BPI, but directed to different host cells,
i.e. bacteria to neutrophils, cell-free LPS to monocytes.
Although the precise molecular determinants of these interactions are
unknown, our findings clearly indicate that recognition by host cells
of BPI-coated endotoxin-containing particles depend both on the
properties of BPI (e.g. the COOH-terminal domain) and on the
nature of the particle itself. The targeting of BPI-coated bacteria and
cell-free LPS to different cells should benefit host defense.
Neutrophils are best equipped to quickly eliminate rapidly multiplying
and disseminating organisms, whereas monocyte-like cells are better
endowed with a digestive apparatus possibly important in detoxification
or antigen presentation (42, 67). It should be noted, however, that the
extent of cellular uptake of BPI:LPSagg, even at relatively
low LPS concentrations (Fig. 10), is limited suggesting the need for
other, as yet unknown, potential targets of BPI:LPSagg
and/or other functional consequences of the delivery of
BPI:LPSagg to monocytes or monocyte-like cells.
The results presented here establish that the carboxyl-terminal domain
is responsible for dictating the fate of the LPS engaged by LBP and
BPI. Both proteins have the ability to interact with LPS to promote the
formation of larger aggregates that are delivered to acceptor molecules
on monocytes. After binding, the fate of the LPS aggregate diverges
depending on the vehicle of delivery. For LBP promoted delivery, the
immediate acceptor molecule is (m)CD14. LPS is transferred via
interaction with the carboxyl-terminal domain of LBP to CD14 which
subsequently activates a receptor molecule, most likely Toll-like
receptor 4, to transmit signals that result in proinflammatory
responses. For BPI promoted delivery, the molecule immediately engaged
upon delivery of LPS is unknown as is the ultimate fate of the
delivered LPS and recipient cells. Whatever the identity of the
acceptor molecule, however, it requires an interaction with the
carboxyl-terminal domain of BPI since similar delivery is not seen with
BPI-21. Studies are in progress to better define the cellular and
subcellular targets of BPI-endotoxin complexes and the host responses
they engender.
We thank Dr. William Nauseef and Janet Hume
for careful reading of the manuscript and Dr. Steve Carroll for
providing recombinant LBP, sCD14, BPI, and rBPI21.
*
This work was supported by United States Public Health
Service Grants DK05472 and PO144642 (to J. W.).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: University of Iowa,
Dept. of Internal Medicine, GH 34W, Iowa City, IA 52242. Tel.: 319-338-0581 (ext. 7534); Fax: 319-339-7162, E-mail:
theresa-gioannini@uiowa.edu.
Published, JBC Papers in Press, December 31, 2001, DOI 10.1074/jbc.M109622200
2
J. Hume and J. Weiss, unpublished observations.
3
D. S. Zhang, A. Teghanemt, J. Weiss, and T. L. Gioannini, unpublished observations.
4
T. Cardozo, unpublished observations.
5
A. Teghanemt and J. Weiss, unpublished observations.
6
N. Iovine, unpublished observations.
The abbreviations used are:
GNB, Gram-negative
bacteria;
BPI, bactericidal/permeability increasing protein;
HBSS+/HBSS
The Carboxyl-terminal Domain of Closely Related Endotoxin-binding
Proteins Determines the Target of Protein-Lipopolysaccharide
Complexes*
,,
**
Department of Medicine, New York University
School of Medicine, New York, New York 10016, the Departments of
Biochemistry, ¶ Microbiology, and § Internal
Medicine, Division of Infectious Diseases, Inflammation Program,
University of Iowa, and Veterans Affairs Medical Center,
Iowa City, Iowa 52242
ABSTRACT
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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
) and spun for 10 min at 30 × g.
The recovered pellet was resuspended in HBSS+ containing 1% HSA.
HSA as indicated: thyroglobulin, blue dextran
(+/HSA), cytochrome c (+/HSA), aldolase, ferritin
(+/HSA).
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES
50% monocytes.
PMN or MNC (0.5-1 × 107/ml) were incubated with
3H-LPS (100 ng/ml) at 37 °C and varying concentrations
of protein. The amount of 3H-LPS that remained associated
after extensive washing of the cells was evaluated. As seen in Fig.
1A, only LBP and
BPIN-LBPC promoted association of
3H-LPS to PMN. BPI and LBPN-BPIC,
as well as BPI-21 (data not shown) were unable to mediate
[3H]LPS binding to PMN at any time tested (up to 2 h). Strikingly different results were obtained when the effect of these
endotoxin-binding proteins on the association of 3H-LPS to
an enriched MNC population was examined (Fig. 1B). In contrast to PMN, both LBP and BPI, as well as both chimeric proteins, BPIN-LBPc and
LBPN-BPIC, promoted association of
3H-LPS with MNC in a dose-dependent manner. LBP
facilitated more 3H-LPS association to MNC than BPI
(>2-fold). BPIN-LBPC and
LBPN-BPIC were nearly equipotent and
intermediate between LBP and BPI in promoting delivery of
3H-LPS to MNC. Maximal 3H-LPS cell association
to PMN and MNC required between 1 and 10 nM
endotoxin-binding protein.
View larger version (18K):
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Fig. 1.
The effect of LBP, BPI, BPI-21, LBP-BPI, and
BPI-LBP on the association of 3H-LPS to PMN and monocyte
enriched-mononuclear cells (MNC). PMN (A) and MNC
(B) were prepared as described under "Experimental
Procedures" and incubated with 100 ng/ml (about 20 nM) of
3H-LPS for 1 h at 37 °C in Hanks', 20 mM HEPES, pH 7.4, containing 1% HSA with varying
concentrations of the indicated endotoxin-binding proteins.
Cell-associated 3H-LPS is plotted as percent of total
3H-LPS added and represents the mean of three or more
experiments ± S.E.
View larger version (16K):
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Fig. 2.
Effect of anti-CD14 antibodies on LBP and BPI
dependent delivery of 3H-LPS to MNC. MNC were
preincubated with either 10 µg/ml MY-4 or MEM-18 for 30 min before
the addition of 3H-LPS (100 ng/ml; about 20 nM)
and the indicated protein (10 nM). Binding was carried out
as described under "Experimental Procedures" and cell-associated
LPS is plotted as percent of total 3H-LPS added. The data
shown represent the mean ± S.E. of three experiments.
View larger version (14K):
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Fig. 3.
Isolation of 3H-LPS aggregates by
Sephacryl S500 HR chromatography. 3H-LPS (List
Biologicals) was reconstituted as described under "Experimental
Procedures" and incubated for 15 min at 37 °C in Hanks', 10 mM HEPES containing 1% HSA before chromatography through
Sephacryl S500 HR using the same buffer. The circled peak
fractions (20-21) were combined and utilized as a source of a
relatively homogeneous population of LPS (LPSagg) as
confirmed by re-chromatography. Data are expressed as percent of total
3H-LPS recovered. Total recovery of 3H-LPS was
>70%.
20 × 106 Da)
(fraction 12, Fig. 4A). The aggregates
contained LBP as shown by immunoblot analysis of isolated
LBP:LPSagg (Fig. 4B). Incubation and
re-chromatography of the purified LBP:LPSagg showed some
minor degree of disaggregation of LPS (fraction 20-24, Fig.
5A). However, treatment of
isolated LBP:LPSagg (fraction 12) with sCD14 produced extensive disaggregation to a complex with apparent mass ~1 × 105 Da (fraction 24-25) as monitored by S500
chromatography (Fig. 5A). The disaggregation of LBP-treated
endotoxin aggregates by incubation with sCD14 is comparable with
results that we have observed previously with endotoxin from
Neisseria meningitidis (5).3
View larger version (31K):
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Fig. 4.
Sephacryl S500 chromatography of purified
LPSagg treated with LBP or BPI. A, isolated
LPSagg (Fig. 3; 100 ng/ml; about 20 nM) was
incubated alone or with 10 nM LBP or BPI in Hanks', 10 mM HEPES, 1% HSA for 15 min at 37 °C before
characterization by gel filtration chromatography. Results shown are
representative of 3 experiments. Data are expressed as percent of
total 3H-LPS recovered. Total recovery of
3H-LPS was >70%. B, immunoblot analysis of
isolated large aggregates recovered after incubation of
LPSagg + LBP: lane 1 represents
recovered peak fraction and lanes 2 and 3 are 0.5 and 1 ng of purified LBP, respectively. C, immunoblot
analysis of isolated large aggregates recovered after incubation of
LPSagg + BPI: lanes 1 represents the recovered
peak fraction and lanes 2 and 3 are 5 and 10 ng
of purified BPI, respectively. Immunoblots with anti-LBP (B)
or anti-BPI (C) antibodies were carried out as described
under "Experimental Procedures." No protein (LBP or BPI) was
detected in the corresponding void volume fractions during
chromatography of purified proteins without LPSagg.
View larger version (16K):
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Fig. 5.
LBP- and sCD14 dependent disaggregation of
3H-LPS as monitored by Sephacryl S500 chromatography.
A, LBP:LPSagg (100 ng of LPS/ml; about 20 nM) recovered after Sephacryl S500 chromatography (fraction
12, Fig. 4A) was incubated for 15 min at 37 °C ± sCD14 (5 µg/ml; about 100 nM) in Hanks', 10 mM HEPES, 1% HSA and then analyzed by gel filtration
chromatography. B, purified LPSagg (100 ng/ml;
about 20 nM) was incubated with LBP (10 nM) ± sCD14 (5 µg/ml; about 100 nM) in
Hanks', 10 mM HEPES, 1% HSA and then analyzed by gel
filtration chromatography. Results shown are representative of 3
experiments. Data are expressed as percent of total 3H-LPS
recovered. Total recovery of 3H-LPS was >70%. The
arrow indicates the peak of elution for LPSagg
alone.
20 × 106 Da) in the presence of BPI (Fig. 6B). Under
the same conditions, incubation of the LPS aggregates with sCD14 alone
had no detectable effect on the aggregation state of LPSagg
(data not shown).
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Fig. 6.
BPI promoted aggregation of
3H-LPS is unaffected by sCD14. A,
BPI:LPSagg (100 ng LPS/ml; about 20 nM)
recovered after Sephacryl S500 chromatography (fraction 12, Fig.
4A) was incubated for 15 min at 37 °C ± sCD14 (5 µg/ml; about 100 nM) in Hanks', 10 mM HEPES,
1% HSA and then analyzed by gel filtration chromatography.
B, purified LPSagg (100 ng/ml; about 20 nM) was incubated with BPI (10 nM) ± sCD14 (5 µg/ml; about 100 nM) in Hanks', 10 mM HEPES, 1% HSA and then analyzed by gel filtration
chromatography. Results shown are representative of 3 experiments.
Data are expressed as percent of total 3H-LPS recovered.
Total recovery of 3H-LPS was >70%. The arrow
indicates the peak of elution for LPSagg alone.
View larger version (16K):
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Fig. 7.
Role of COOH-terminal domain of LBP in
sCD14-dependent disaggregation of LPS.
3H-LPSagg (100 ng/ml; about 20 nM)
was treated with 10 nM BPIN-LBPC or
LBPN-BPIC and sCD14 (5 µg/ml; about 100 nM) for 15 min at 37 °C and analyzed by chromatography
on Sephacryl S500. Results shown are representative of 3 experiments.
Data are expressed as percent of total 3H-LPS recovered.
Total recovery of 3H-LPS was >70%. The arrow
indicates the peak of elution for LPSagg alone.
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Fig. 8.
Role of COOH-terminal domain of LBP in
sCD14-independent disaggregation of LPS in the presence of EDTA.
LPSagg (100 ng/ml; about 20 nM) ± 10 nM LBP or LBPN-BPIC (A)
or BPI or BPIN-LBPC (B) was
incubated for 15 min at 37 °C ± in Hanks' without
Mg2+ or Ca2+, 10 mM HEPES, 5 mM EDTA, 1% HSA and then analyzed by gel filtration
chromatography on Sephacryl S500. Data are expressed as percent of
total 3H-LPS recovered. Total recovery of
3H-LPS was >70%.
View larger version (17K):
[in a new window]
Fig. 9.
Effect of LBP and BPI on activation of MNC by
3H-LPS as measured by lucigenin-enhanced
chemiluminescence. 3H-LPS (100 ng/ml; about 20 nM) was preincubated with LBP (A) or BPI
(B) for 1 h, 37 °C in HBSS+, 10 mM
HEPES, 1% HSA before addition to MNC (0.5-1 × 107/ml) plus 30 µM lucigenin. Results are
expressed as chemiluminescence units (CLU, i.e.
photons per second). The data shown correspond to results from a
representative experiment where each point represents the average of
duplicate samples. At least three independent experiments were carried
out.
20 × 106 Da) isolated by gel filtration
chromatography (Fig. 4). The isolated aggregates are free of any LBP or
BPI unassociated with LPSagg precluding any possible
effects of free protein on the functional behavior of the aggregates.
The LBP- and BPI- coated LPSagg associated with MNC far
more than protein-free LPSagg (Fig.
10A). Increased cell
association of LBP:LPSagg, was accompanied by increased
cell activation (Fig. 10B) whereas binding of
BPI:LPSagg provoked little cell activation (Fig.
10B). These findings clearly demonstrate the different
functional consequences of the binding of LBP:LPSagg versus BPI:LPSagg to monocytes.
View larger version (12K):
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Fig. 10.
Interaction with and activation of MNC by
purified LBP:LPSagg and
BPI:LPSagg. Purified LPSagg (see
Fig. 3) and purified LBP:LPSagg and BPI:LPSagg
(see Fig. 4) were incubated with MNC to measure cell association of
3H-LPS (A) and lucigenin-enhanced
chemiluminescence (B) as described under "Experimental
Procedures" and legends to Figs. 1 and 9 except that 60 µM lucigenin was used. The concentration of
3H-LPS added in panel A was about 3 ng; doses
used in panel B are indicated. Chemiluminescence induced by
addition of a column fraction containing no 3H-LPS or
column buffer alone was <1000 chemiluminescence units and was
subtracted from values obtained in the LPS containing samples. The
results shown represent the mean of two or three independent
experiments, each in duplicate.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 × 106 Da.
Estimates of the amount of BPI and LBP bound to the LPSagg indicate that the bound protein accounts for <30% of the apparent increase in size suggesting additional alterations in the aggregation state of LPS. BPI-induced alterations in LPS density and light scattering have been previously observed (47) that could reflect similar physical alterations. However, many earlier studies have observed direct disaggregating effects of LBP on LPS (8, 19, 47, 48,
50). These experiments typically lacked divalent cations
(i.e. Mg2+ and Ca2+) needed to
maintain the close intermolecular packing of the highly anionic LPS
(51-53). By comparing the effects of LBP in the presence (Figs. 4 and
5) and absence (Fig. 8) of divalent cations, we have directly
demonstrated that when physiological extracellular levels of
Mg2+ and Ca2+ are present, efficient
disaggregation of LPS by LBP is only possible when (s)CD14 is also
present (Figs. 4 and 5) (5). Both sCD14-dependent disaggregation of LPS in the presence of Mg2+ and
Ca2+ and sCD14-independent disaggregation of LPS in the
presence of EDTA are promoted by the COOH-terminal domain of LBP (Figs.
7 and 8). Together with the role of this domain of LBP in delivery of
LPS aggregates to CD14 (Fig. 2), these findings suggest that the
COOH-terminal domain of LBP may facilitate formation of small bioactive
endotoxin-CD14 complexes by helping to destabilize LPS aggregates and
promote contact with CD14. The ability of isolated LBP:LPSagg to interact with sCD14 (Fig. 5A) and
cells containing mCD14 (Fig. 10A) in the same manner as the
addition of a mixture of LPSagg and LBP indicates that
these LBP:LPSagg represent functionally relevant intermediates.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, Hanks' balanced salt solution +/ Mg2+ and
Ca2+;
HSA, human serum albumin;
LBP, lipopolysaccharide-binding protein;
LPS, lipopolysaccharide;
LPSagg, lipopolysaccharide aggregates;
PMN, polymorphonuclear leukocytes;
mAb, monoclonal antibody;
MNC, monocyte-enriched cells;
s/m CD14, soluble CD14 or membrane CD14.
REFERENCES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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