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Infection and Immunity, December 2000, p. 6519-6525, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Lactoferrin Interacts with Soluble CD14 and Inhibits
Expression of Endothelial Adhesion Molecules, E-Selectin and
ICAM-1, Induced by the CD14-Lipopolysaccharide Complex
S.
Baveye,1
E.
Elass,1
D. G.
Fernig,2
C.
Blanquart,1
J.
Mazurier,1 and
D.
Legrand1,*
Laboratoire de Chimie Biologique et Unité Mixte de
Recherche n°8576 du Centre National de la Recherche Scientifique,
Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France,1 and
School of Biological Sciences, Life Sciences Building,
University of Liverpool, Liverpool L69 7ZB, United
Kingdom2
Received 22 February 2000/Returned for modification 15 May
2000/Accepted 8 September 2000
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ABSTRACT |
Lipopolysaccharides (LPS), either in the free form or complexed to
CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin
(Lf), a LPS-chelating glycoprotein, protects animals against septic
shock. Since optimal protection requires administration of Lf prior to
lethal doses of LPS, we hypothesized that interactions between Lf and
soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf
(hLf) were used to determine the kinetic binding parameters of hLf to
free sCD14 in an optical biosensor. The results demonstrated that hLf
bound specifically and with a high affinity (Kd = 16 ± 7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with
different binding properties, with sCD14 complexed to LPS or lipid
A-2-keto-3-deoxyoctonic acid-heptose. In a second step, we have
investigated whether the capacity of hLf to interact with sCD14 could
modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by
the sCD14-LPS complex on human umbilical vein endothelial cells
(HUVEC). Our experiments show that hLf significantly inhibited both
E-selectin and ICAM-1 expressions at the surface of HUVEC. In
conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to
chelate LPS but also to its ability to interact with sCD14 and with the
sCD14 complexed to LPS, thus modifying the activation of endothelial cells.
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INTRODUCTION |
One of the central proinflammatory
functions of endothelial cells is the recruitment of circulating
leukocytes at inflammatory tissue sites. Lipopolysaccharides (LPS)
derived from Gram-negative bacteria are potent stimulators of
inflammation (34, 43) that induce either directly or through
the intermediary of cytokines (11), the expression of
adhesion molecules such as endothelial-leukocyte adhesion molecule 1 (E-selectin) and intercellular adhesion molecule 1 (ICAM-1) (7,
36). Endotoxin stimulation of endothelial cells is mediated by
soluble CD14 (sCD14), a specific LPS receptor (3, 18, 19,
36). CD14 is a 55-kDa glycoprotein that exists both as a soluble
protein found in serum at concentrations of 2 to 6 µg/ml
(16) and as a glycosylphosphatidylinositol-anchored protein
(mCD14) on the surface of monocytes-macrophages (5, 50, 52).
At low endotoxin levels, a serum acute protein called the LPS-binding
protein (LBP), which catalyzes the transfer of LPS monomers from
aggregates to CD14, enhances the sensitivity of cells to LPS (19,
36, 44). Nevertheless, at high LPS concentrations, LBP is not
essential to the activation of endothelial cells and LPS may directly
bind to CD14 to form an sCD14-LPS complex (18, 19, 42).
Thus, the activation of endothelial cells by the sCD14-LPS complex
promotes leukocyte infiltration and microvascular thrombosis and
contributes, during septic shock, to the pathogenesis of disseminated
intravascular inflammation. This phenomenon leads to severe damage of
endothelium (8). Various LPS-binding proteins modulate the
activation of cells (45), among which is lactoferrin (Lf),
an iron-binding glycoprotein found in exocrine secretions of mammals
and released from granules of neutrophils during inflammation (31). Following infection, Lf concentrations higher than 20 µg/ml can be detected in blood (6). Interactions between
Lf and LPS have been thoroughly investigated. Human Lf (hLf) binds to
the lipid A region of LPS with a high affinity (2).
Experiments using hLf variants and mutants demonstrated that amino acid
residues 1 to 5 and 28 to 34 of hLf interact with Escherichia
coli LPS (14, 17). In vitro, Lf prevents the
LBP-mediated binding of LPS to mCD14 (13) and decreases the
release of cytokines such as interleukin 1 (IL-1), IL-6, and tumor
necrosis factor alpha from LPS-stimulated monocytes (10,
33). Lf might also modulate the inflammatory process in vivo.
Indeed, studies reported the protective function of Lf against
sublethal doses of LPS in mice (29, 51). Recently, the
protective effect of Lf feeding against endotoxin lethal shock in
germfree piglets has been described (25). These observations
indicate that Lf is one of the key molecules which modulates the
inflammatory responses (4).
The ability of Lf to bind free LPS may account, in part, for the
anti-inflammatory activities of the protein. However, since optimal
protection of animals against the septic shock requires a 12- to 24-h
preinjection of Lf, it may be assumed that other mechanisms are
involved. We hypothesized that interactions between Lf and LPS
receptors such as sCD14 exist.
In this study, we analyzed the potential protective effect of Lf under
LBP-independent septic shock conditions. We first studied the binding
of various concentrations of sCD14 to hLf with an optical biosensor.
Affinity chromatography was then used to study the binding of sCD14 to
hLf in the presence of E. coli 055:B5 LPS and LPS moieties.
Lastly, we investigated whether hLf modifies the activating properties
of the sCD14-LPS complex on endothelial cells. For this purpose, the
effect of hLf on the expression of E-selectin and ICAM-1 induced by the
sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC) was determined.
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MATERIALS AND METHODS |
Reagents.
RPMI 1640 medium was obtained from Gibco-BRL
(Eragny, France) and endothelial cell growth medium SFM supplemented
with fetal calf serum, endothelial cell growth supplement, heparin,
epidermal growth factor, bovine fibroblast growth factor,
hydrocortisone, gentamicin, and amphotericin B was from PromoCell
(Heidelberg, Germany). Both biotin hydrazide and Ultralink hydrazide
were from Pierce Chemicals Co. (Rockford, Ill.). SP-Sepharose fast flow column and PD10 G-25 column were from Pharmacia (Uppsala, Sweden). The
apyrogen water was from Cooper (Melun, France), the Centricon-30 ultrafiltration units were from Amicon (Danvers, Mass.), and the nitrocellulose was from Schleicher & Schuell (Dassel, Germany). Dulbecco's phosphate-buffered saline (PBS), human serum transferrin (hTF), bovine serum albumin, collagenase, gelatin, diaminobenzidine peroxidase substrate tablet set,
o-phenylenediamine-dihydrochloride, and LPS O55:B5 from
E. coli were purchased from Sigma Chemical Co. (St. Louis,
Mo.). Lipid A-2-keto-3-deoxyoctonic acid (KDO)-heptose and lipid A
were purified from a rough mutant (395MR10, Rd chemotype) of
Salmonella enterica serovar Typhimurium as previously
described (32), and the purity of preparations was checked
by mass spectral analysis. These LPS fractions were generous gifts from
I. Mattsby-Baltzer (Department of Clinical Bacteriology,
Göteborg, Sweden). Recombinant human sCD14 was purchased from
Biometec (Greifswald, Germany). It was obtained from serum-free culture
supernatant of CHO cells transfected with human CD14 cDNA cloned into
pPOL-DHFR expression vector (41). Rabbit anti-CD14
polyclonal antibodies were purchased from Biometec, goat
peroxidase-labeled anti rabbit immunoglobulin G (IgG) from Biosys
(Compiègne, France), mouse monoclonal anti-ICAM-1 antibodies
(clone 15.1) from Tebu (Le Perray-en-Yvelines, France), mouse
monoclonal anti-E-selectin antibodies (clone 1.2B6) from Immunotech
(Marseille, France), peroxidase-conjugated sheep anti mouse IgG from
Sanofi (Marnes-la-Coquette, France) and isotype control IgG1 from Sigma
Chemical Co.
Endothelial cell culture.
Endothelial cells (HUVEC) were
derived from human umbilical vein, according to the method previously
described (21). Briefly, after treatment of umbilical vein
with 0.2% (wt/vol) collagenase in 37°C-prewarmed RPMI for 30 min,
HUVEC were collected by centrifugation (600 × g for 15 min). Cells were resuspended in endothelial cell growth medium SFM and
cultured in gelatin-coated 35-mm-diameter tissue culture wells at
37°C and 5% CO2. They were collected after trypsinization and then cultured in gelatin-coated 96-well
flat-bottomed culture plates until confluency. Only cells of the third
and the fourth passages were used. Viability was over 96% as
determined by trypan blue dye exclusion.
Preparation of LPS-free hLf.
Native hLf was purified from
fresh human milk by cation-exchange chromatography and iron saturated,
as previously reported (35, 39). Homogeneity of the protein
was checked by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Since the interaction between LPS and Lf
was abrogated by NaCl concentrations higher than 0.4 M (46),
50 mg of purified hLf was injected on a 7-by-1-cm SP-Sepharose fast
flow column equilibrated in 0.1 M NaCl and then washed with 70 ml of
0.5 M NaCl. hLf was eluted with 2 M NaCl and desalted on a PD10 G-25
column equilibrated in 0.1 M NaCl. All buffers were prepared with
pyrogen-free water. The LPS contamination of these hLf fractions was
less than 50 pg of endotoxin/mg of protein, as estimated by the
Limulus amoebocyte lysate assay (QCL1000; BioWhittaker,
Walkersville, Md.).
Preparation of biotin-labeled hLf.
To avoid possible steric
hindrance of the interactions of the hLf polypeptide with sCD14, we
labeled hLf through its glycan moiety after mild periodate oxidation of
N-acetylneuraminic acid residues. This method has been
successfully carried out to label Lf without affecting its biological
activity (26, 28). All solutions were prepared with
pyrogen-free water. The glycan moiety of hLf was biotinylated by
coupling biotin hydrazide to aldehyde groups produced by mild periodate
oxidation of N-acetylneuraminic acid residues. Briefly, hLf
(5 mg) dissolved in 230 µl of 0.1 M sodium acetate-0.15 M NaCl (pH
5.6) was mixed with 100 µl of 0.018 M sodium periodate and incubated
for 10 min at 4°C. The reaction was stopped by adding 10 µl of
ethylene glycol and desalted on a Sephadex G-25 PD10 column in PBS.
Oxidized hLf was then incubated with biotin hydrazide (5 mg in 3 ml of
PBS) for 2 h at room temperature with gentle mixing. Free biotin
hydrazide was removed through Centricon-30 filters. After concentration
of labeled protein to a final volume of 500 µl, biotinylated hLf was
passed through a Sephadex PD10 G-25 column in PBS. LPS contamination of
the labeled hLf was controlled. Biotinylated hLf was used for the
biosensor studies.
Analysis of sCD14 binding to hLf in an optical biosensor.
Binding reactions were carried out in an IAsys two-channel resonant
mirror biosensor at 20°C (Affinity Sensors, Saxon Hill, Cambridge,
United Kingdom) (37, 38) with minor modifications. Planar
biotin surfaces, with which a signal of 600 arc s corresponds to 1 ng
of bound protein/mm2, were derivatized with streptavidin
according to the manufacturer's instructions. Controls showed that
sCD14 did not bind to streptavidin-derivatized biotin surfaces (result
not shown). Biotinylated hLf was immobilized on planar
streptavidin-derivatized surfaces, which were then washed with PBS. The
distribution of the immobilized hLf and of the bound sCD14 on the
surface of the biosensor cuvette was inspected by the resonance scan,
which showed that at all times these molecules were distributed
uniformly on the sensor surface and therefore were not microaggregated.
Binding assays were conducted in a final volume of 30 µl of PBS at
20 ± 0.1°C. The ligate was added at a known concentration in 1 µl to 5 µl of PBS to the cuvette to give a final concentration of
sCD14 ranging from 14 to 73 nM. To remove residual bound ligate after
the dissociation phase, and thus regenerate the immobilized ligand, the
cuvette was washed three times with 50 µl of 2 M NaCl-10 mM
Na2HPO4, pH 7.2, and three times with 50 µl
of 20 mM HCl. Data were pooled from experiments carried out with
different amounts of immobilized hLf (0.2, 0.6, and 1.2 ng/mm2). For the calculation of kon,
low concentrations of ligate (sCD14) were used, whereas for the
measurement of koff, higher concentrations of
ligate were employed (1 µM) to avoid any rebinding artifacts. The
binding parameters kon and
koff were calculated from the association and
dissociation phases of the binding reactions, respectively, using the
nonlinear curve-fitting FastFit software (Affinity Sensors) provided
with the instrument. The dissociation constant
(Kd) was calculated from the association and
dissociation rate constants and from the extent of binding observed
near equilibrium.
Affinity chromatography studies.
Purified hLf and human
serum transferrin were immobilized on Ultralink hydrazide gel according
to manufacturer's instructions and used to study the binding of human
recombinant sCD14. Two milligrams of protein was bound per ml of
Ultralink hydrazide gel.
Two micrograms of sCD14 was preincubated in the absence or in the
presence of an excess of
E. coli O55:B5 LPS (10 µg in 200
µl of PBS) for 1 h at 37°C and further incubated for 3 h
at 37°C
with 50 µg of hLf immobilized on the Ultralink hydrazide
gel.
As reported elsewhere (
17), such experimental
conditions led
to full complexation of sCD14 to LPS in the absence of
LBP. Prior
to use, LPS suspensions were sonicated and diluted in
Dulbecco's
PBS without Ca
2+ and Mg
2+ to avoid
aggregation of LPS molecules. Similar experiments were
also performed
with lipid A and lipid A-KDO-heptose from serovar
Typhimurium.
Nonspecific binding of sCD14 was estimated on uncoupled
Ultralink
Hydrazide gel. A control was performed with immobilized
hTf. The gel
was collected by centrifugation at 600 ×
g for 5
min
and washed with 10 ml of PBS. The sCD14 bound to the gel was
sequentially eluted with 3 volumes of 200 µl of 0.5 M NaCl in
20 mM
sodium phosphate buffer, pH 7.4, 3 volumes of 200 µl of
1 M NaCl in
this buffer, 2 volumes of 200 µl of 0.2 M glycine-HCl
(pH 2.3)
containing 0.5% (vol/vol) Triton X-100, and 300 µl of
SDS (10%,
wt/vol). Polypeptides in 100 µl of each fraction were
separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
in 7.5% (wt/vol)
acrylamide gels and then transferred to a 0.45-µm-pore-size
nitrocellulose membrane. The membranes were soaked in PBS containing
2% (wt/vol) gelatin for 90 min and then incubated for 2 h with
an
antiserum to human CD14 (1:1,500 dilution in PBS containing
0.05%
[vol/vol] Tween 20). The membranes were washed three times
with PBS
containing 0.1% (vol/vol) Tween 20 and then incubated
for 1 h
with goat peroxidase-labeled anti-rabbit IgG (1:1,000
dilution in PBS
containing 0.05% [vol/vol] Tween 20). Immunoreactive
sCD14 was
detected with the diaminobenzidine peroxidase substrate
tablet set. All
immunochemical staining steps were performed at
room
temperature.
Expression of ICAM-1 and E-selectin on HUVEC.
Endothelial
ICAM-1 and E-selectin expressions were measured by enzyme immunoassay,
as previously described (20, 36, 49). Cells were plated into
gelatin-coated 96-well tissue culture plates and grown to confluence.
To study the ICAM-1 expression, cells were washed twice with RPMI and
incubated for 24 h at 37°C in 5% CO
2, either with
E. coli O55:B5 LPS (100 ng/ml) or with a mixture of LPS (100 ng/ml)
and sCD14 (2 µg/ml) preincubated for 30 min at room
temperature.
Controls were performed without LPS and without sCD14,
with sCD14
and without LPS, with hLf alone, and with hLf and sCD14. As
previously
described (
24) the maximal ICAM-1 expression was
obtained after
24 h of incubation with cells. The effect of hLf on
the expression
of ICAM-1 was investigated under conditions similar to
those described
above. Before incubation with cells, hLf (50 µg/ml)
was preincubated
for 30 min at room temperature with LPS in the
presence of sCD14.
In some experiments, the hLf-sCD14, LPS-sCD14, or
LPS-hLf mixtures
were incubated for 30 min at room temperature before
the addition
of LPS, hLf, or sCD14, respectively, and then added for
24 h with
cells at 37°C. The medium was removed, and the cells
were washed
twice with PBS. HUVEC monolayers were fixed at room
temperature
for 15 min with 2% paraformaldehyde. The fixative was
removed
and replaced with 100 mM glycine to block reactive aldehyde
groups.
After two washes with PBS, plates were incubated for 30 min at
37°C with 2% (wt/vol) bovine serum albumin. Then, anti-mouse ICAM-1
monoclonal antibody (4 µg/ml) was added and incubated at 37°C
for
1 h. After washing, peroxidase-conjugated sheep anti-mouse
IgG
diluted 1:2,000 in PBS was added for 30 min at 37°C. An isotype
control (IgG1) was used to evaluate the nonspecific binding of
the
monoclonal antibody. Staining was achieved by adding 150 µl
of
o-phenylenediamine-dihydrochloride per well for 20 min at
room
temperature, according to manufacturer's instructions. The
reaction
was stopped with 50 µl of 2 M H
2SO
4
per well, and the absorbance
at 490 nm was measured on an enzyme-linked
immunosorbent assay
plate
reader.
To study the endothelial E-selectin expression, the protocol used was
the same as that described above for ICAM-1, but the
incubation with
the cells was only 5 h at 37°C and the concentration
of LPS used
was 1 µg/ml. Moreover, mouse monoclonal anti-ICAM-1
antibody was
replaced by a mouse monoclonal anti-E-selectin antibody
at a
concentration of 4 µg/ml.
Statistical analysis.
Data are presented as the mean ± standard error (SE) for the indicated number of independent
experiments. Statistical significance was analyzed by Student's
t test for unpaired data. Values of P < 0.05 were considered to be significant.
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RESULTS |
Analysis of sCD14 binding to hLf in an optical biosensor.
The
association phase of the binding reaction between sCD14 and hLf was
fairly rapid (Fig. 1). Analysis of the
binding curves from four experiments indicated that the binding of
sCD14 to hLf was saturable and monophasic (Fig. 1 and Table
1). The association rate constant
(kass) and dissociation rate constant
(kdiss) for the sCD14-hLf interaction were
360,000 ± 110,000 M
1 s1 and
0.0058 ± 0.0017 s1, respectively (Table 1). The
equilibrium dissociation constant (Kd)
calculated from the ratio of the kinetic rate constants
(kdiss/kass) was 16 ± 7 nM. The Kd calculated from the extent of
binding observed near equilibrium was 45 ± 30 nM, a value which
was similar to that calculated from kinetic parameters. These results
demonstrate that sCD14 binds to hLf with a high affinity and with
fairly fast kinetics.
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FIG. 1.
Analysis of sCD14 binding to hLf in an optical
biosensor. Biotinylated hLf was immobilized on a
streptavidin-derivatized biotin surface as described in Materials and
Methods. The binding of different concentrations of sCD14 (14 to 73 nM)
to immobilized hLf was monitored in real time for about 300 s.
Four independent sets of binding reactions were performed, of which one
is presented. The inset shows that a plot of kon
against ligand concentration yields a straight line (r = 0.989), the slope of which corresponds to
kass. The kon of sCD14
for hLf at each concentration of sCD14 was determined using the FastFit
software as described in Materials and Methods.
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Similar binding experiments were performed with the complex of sCD14
and
E. coli O55:B5 LPS. However, the addition of free
LPS in
PBS to the cuvette induced a negative bulk shift (10 arc
s), suggesting
that the refractive index of a solution containing
LPS in PBS is lower
than that of PBS alone, in contrast to the
positive bulk shift observed
with proteins (
37,
38). Since
excess LPS attenuated the
response of the optical biosensor, the
binding parameters of sCD14 to
hLf in the presence of LPS could
not be determined. Therefore, the
binding of sCD14 to hLf in the
presence of LPS and LPS moieties was
assessed by affinity chromatography
on Ultralink hydrazide
gel-immobilized
hLf.
Binding of sCD14 to immobilized hLf in the presence of LPS and LPS
moieties.
Human sCD14 alone or in the presence of E. coli O55:B5 LPS was incubated with Ultralink hydrazide
gel-immobilized hLf and then sequentially eluted by solutions
containing 0.5 and 1 M NaCl, glycine-HCl (pH 2.3), 0.5% (vol/vol)
Triton X-100, and 10% (wt/vol) SDS. Figure
2A shows that free sCD14 only dissociated
from hLf under stringent conditions such as pH 2.3, Triton X-100, and
especially SDS treatment. No sCD14 was detected in the NaCl fractions.
This result ties well with the high-affinity interactions detected by
the biosensor technique. When sCD14 was incubated with excess LPS prior to affinity chromatography, comparable amounts of sCD14 bound to hLf, but dissociation occurred under milder conditions (Fig.
2B). As a matter of fact, sCD14 mainly eluted from the hLf gel in the
first 0.5 M NaCl fraction. Only traces of sCD14 eluted in the pH 2.3 fraction. This result suggests that the sCD14-LPS complex did bind to
hLf but through labile interactions. No nonspecific binding of sCD14 to
the uncoupled Ultralink hydrazide gel was detected (data not shown).
Likewise in a control performed with hTf immobilized in a manner
identical to that employed for hLf, CD14 eluted in the PBS wash steps
and not in the NaCl, acidic, or detergent fractions (data not shown).
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FIG. 2.
Affinity chromatography of sCD14 to immobilized hLf in
the absence of LPS (A) or in the presence of LPS (B), lipid A (C), or
lipid A-KDO-heptose (D). sCD14 was preincubated 1 h with or
without the different LPS moieties, and affinity chromatography was
performed on hLf bound to Ultralink hydrazide gel as described in
Materials and Methods. After 3 h of incubation, the gels were
washed three times with 20 mM sodium phosphate, pH 7.4, buffers
containing 0.5 M and 1 M NaCl, twice with a 0.2 M glycine-HCl, pH 2.3, buffer containing 0.5% (vol/vol) Triton X-100 (Gly/HCl), and once with
300 µl of SDS (10%, wt/vol). Identical volumes of the corresponding
washing solutions were subjected to SDS-PAGE (7.5% polyacrylamide) and
transferred to nitrocellulose. Immunostaining was performed with
specific anti-CD14 polyclonal antibodies. Lanes labeled 1, 2, and 3 correspond to the successive washes with each buffer.
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In order to investigate the region of LPS responsible for weaker
saline-labile interactions between the sCD14-LPS complex
and hLf,
experiments were performed with both lipid A and lipid
A-KDO-heptose
(LPS inner core) moieties. Figure
2C shows that
the presence of lipid A
on sCD14 only slightly altered its binding
to hLf, since sCD14 was
detected in equal amounts in the first
pH 2.3 and SDS fractions but not
in the NaCl fractions. In the
presence of the lipid A-KDO-heptose core,
intermediate elution
features were observed (Fig.
2D). As a matter of
fact, sCD14 was
mainly found in the first pH 2.3 fraction, but
substantial amounts
were also detected in the first NaCl 0.5 M, the
second pH 2.3,
and the SDS fractions. Our results indicate that the
size (and
nature) of the LPS moiety influenced the binding
characteristics
of sCD14 to
hLf.
Effect of hLf on expression of endothelial ICAM-1 and E-selectin
induced by the sCD14-LPS complex.
The capacity of hLf to modulate
the expression of E-selectin and ICAM-1 induced by the sCD14-LPS
complex on HUVEC was assessed in enzyme immunoassays using E-selectin
and ICAM-1 antibodies (20, 36, 49). LPS concentrations
ranging from 10 ng/ml to 10 µg/ml were used to trigger the expression
of ICAM-1 and of E-selectin. Since maximal expressions were gained with
100 ng/ml and 1 µg/ml LPS for ICAM-1 and E-selectin, respectively
(data not shown), these concentrations were used in further experiments.
As shown in Fig.
3, HUVEC exhibited a low
basal expression of ICAM-1, which was not significantly increased in
the presence
of sCD14, hLf, or both proteins (negative controls) or
LPS. When
compared to unstimulated cells, a fivefold-higher level of
ICAM-1
was detected in the presence of sCD14-LPS (
P < 0.05). This demonstrated
that the sCD14-LPS complex induced the
expression of ICAM-1 on
endothelial cells. Interestingly, the ICAM-1
expression induced
by the sCD14-LPS complex on HUVEC was significantly
decreased
in the presence of hLf (50 µg/ml). Taking the expression
level
induced by sCD14-LPS as a reference, a 51% ± 2% inhibition of
the ICAM-1 expression was calculated when hLf was incubated with
sCD14
and LPS at the same time (
P < 0.05). This inhibition
was
similar to that detected when hLf was mixed with LPS 30 min prior
to sCD14 (56% ± 14%) or when an sCD14-LPS complex was preformed
before the addition to hLf (53% ± 19%) (
P < 0.05).
A higher inhibition
(81% ± 5%) was measured when LPS was added after
the preincubation
of sCD14 with hLf.
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FIG. 3.
Effect of hLf on the LPS-induced ICAM-1 expression on
HUVEC. Various mixtures were preincubated for 30 min at room
temperature before 24 h of incubation at 37°C with cells (per
milliliter) as follows: 2 µg of sCD14 (sCD14); 50 µg of hLf alone
(hLf) or with 2 µg of sCD14 (hLf + sCD14); 100 ng of LPS alone
(LPS) or with 2 µg of sCD14 (sCD14 + LPS); 50 µg of hLf with
100 ng of LPS (hLf + LPS); or 2 µg of sCD14, 100 ng of LPS, and
50 µg of hLf (sCD14 + LPS + hLf). In some cases, the
sCD14-LPS, LPS-hLf, and sCD14-hLf mixtures were preincubated for 30 min
at room temperature prior to addition of hLf [(sCD14 + LPS) + hLf], sCD14 [(LPS + hLf) + sCD14], and LPS [(sCD14 + hLf) + LPS], respectively, and further incubation with cells. A
control was performed with cells in the absence of hLf, sCD14, and LPS
(none). The expression of ICAM-1 on HUVEC was estimated by enzyme
immunoassay as described in Materials and Methods. Results are
expressed as mean values of optical density at 490 nm (O. D. 490 nm) ± SE (error bars) from quadruplicates, after subtracting
nonspecific binding of antibodies, and are representative of at least
two separate experiments conducted with HUVEC isolated from human
umbilical veins from different donors.
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As shown in Fig.
4, similar results were
obtained with the E-selectin expression on HUVEC. Indeed, two- and
fivefold-higher
levels of E-selectin were detected in the presence of
LPS and
sCD14-LPS, respectively. hLf had no effect on the low
activation
level of cells obtained with LPS in the absence of sCD14. In
contrast,
the inhibition of the expression induced by sCD14-LPS in the
presence
of hLf was 48% ± 9% when hLf was incubated with sCD14 and
LPS
at the same time (
P < 0.05), 50% ± 13% when hLf
was mixed with
sCD14 30 min prior to LPS (
P < 0.05),
52% ± 17% when an sCD14-LPS
complex was preformed before the
addition of hLf, and 38% ± 6%
when sCD14 was added after the
preincubation of hLf with LPS.
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FIG. 4.
Effect of hLf on the LPS-induced E-selectin expression
on HUVEC. Various mixtures were preincubated for 30 min at room
temperature before 5 h of incubation at 37°C with cells (per
milliliter) as follows: 2 µg of sCD14 (sCD14); 50 µg of hLf alone
(hLf) or with 2 µg of sCD14 (hLf + sCD14); 1 µg of LPS alone
(LPS) or with 2 µg of sCD14 (sCD14 + LPS); 50 µg of hLf with 1 µg of LPS (hLf + LPS); or 2 µg of sCD14, 1 µg of LPS, and 50 µg of hLf (sCD14 + LPS + hLf). In some cases, the
sCD14-LPS, LPS-hLf, and sCD14-hLf mixtures were preincubated for 30 min
at room temperature prior to addition of hLf [(sCD14 + LPS) + hLf], sCD14 [(LPS + hLf) + sCD14], and LPS [(sCD14 + hLf) + LPS], respectively, and further incubation with cells. A
control was performed with cells in the absence of hLf, sCD14, and LPS
(none). The expression of E-selectin on HUVEC was estimated by enzyme
immunoassay as described in Materials and Methods. Results are
expressed as mean values of optical density at 490 nm (O. D. 490 nm) ± SE (error bars) from quadruplicates, after subtracting
nonspecific binding of antibodies, and are representative of at least
four separate experiments conducted with HUVEC isolated from human
umbilical veins from different donors.
|
|
These findings suggest that hLf modulates the expressions of
endothelial ICAM-1 and E-selectin through its interaction with
sCD14
and the sCD14-LPS
complex.
| |
DISCUSSION |
The protective effect of Lf against endotoxin lethal shock in mice
or in germfree piglets was reported (25, 29, 51). The
endotoxin-chelating properties of Lf and its ability to compete with
LBP for LPS binding (13) explain in part the role of the protein in the modulation of inflammation (2, 13, 14). Indeed, Lf prevents the release of cytokines induced by LPS from monocytes in vitro (10, 33) and in vivo (29, 51).
However, the optimal protection of animals against induced septicemia
requires a 12- to 24-h preinjection of Lf (51), which
suggests that this protein may act by mechanisms in addition to simple
LPS scavenging. We hypothesized that interactions between hLf and LPS
receptors such as sCD14 exist and thus interfere with the activation of target cells. The aim of this study was to investigate the potential protective effect of lactoferrin under septic shock conditions. Since
LBP is not essential at high LPS concentrations and in order to focus
on potential interactions between hLf and sCD14, LBP was not included
in the experiments.
In the present report, we provide evidence that sCD14 binds
specifically to hLf with a high affinity (Kd = 16 ± 7 nM). Basic sequences 2RRRR5
and 28RKVRGPP34, which are close and accessible
at the surface of hLf (1), interact with various anionic
molecules such as heparin (9), proteoglycans
(27), and LPS (23, 48) (Fig.
5). In sCD14, acidic residues
35AVEVE39 and
53RVDADADPRQY63 are involved in the
interactions with LPS (23, 48), while amino acids
7ELDDEDF13 are essential for cell activation
(22, 40) (Fig. 5). We postulate that all or part of the
basic or acidic amphipatic stretches present in hLf and sCD14 are
responsible for the high-affinity interactions between these two
glycoproteins, resulting in the formation of a stable sCD14-hLf
complex. The use of mutated recombinant hLf and sCD14 will gain further
insight into the importance of these stretches in the interactions.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 5.
N-terminal sequences of human sCD14 and hLf. The
underlined amino acids in the sCD14 sequence represent the cell
signaling site (22, 40), and the residues in boldface type
are those implicated in the LPS binding (23, 48). The amino
acids in boldface type in the hLf sequence are the two N-terminal basic
stretches involved in the interactions of hLf with LPS (2, 13, 14,
47).
|
|
Since Biosensor technology failed in determining the binding parameters
of sCD14 to hLf in the presence of E. coli O55:B5 LPS,
comparative affinity chromatography studies were undertaken. Our
results show that the sCD14-LPS complex bound to hLf but through more-labile interactions than that between sCD14 and hLf. As a matter
of fact, while sCD14 alone mainly dissociated from hLf under drastic
conditions (SDS), its binding to hLf in the presence of LPS was mainly
disrupted in the first NaCl 0.5 M wash. This phenomenon may be relevant
to the way that hLf binds to the sCD14-LPS complex. In fact, it is not
known whether hLf binds to the sCD14 or to the LPS counterpart of the
complex. If hLf binds to the sCD14 moiety, it is likely that the
presence of LPS impedes further interactions of hLf with the
LPS-binding residues 35 to 39 and 53 to 63 (23, 48) of
sCD14. Nevertheless, the basic region of hLf could still bind to the
acidic stretch 7ELDDEDF13 of sCD14,
thus generating saline-labile interactions. The ability of LPS moieties
to weaken sCD14-hLf interactions was also observed but to a lower
extent and in an LPS moiety size-dependent way. Lipid A, the smallest
moiety, only slightly interfered in the binding of hLf to sCD14. The
binding properties of LPS moieties to sCD14, which are not as well
defined as those of LPS, are probably responsible for this difference.
In particular, it is not known whether the LPS moieties bind to one,
two, or none of the sCD14 LPS-binding stretches, residues 35 to 39 and
53 to 63, which could thus remain partially accessible to hLf.
Furthermore, it may be assumed that the affinities of sCD14 for either
lipid A or lipid A-KDO-heptose are lower than that for LPS (34 nM
[46]) or hLf (16 ± 7 nM). Therefore, hLf could
displace the LPS moieties from sCD14.
Another possibility is the binding of hLf to the LPS moiety of the
sCD14-LPS complex. It was recently reported that sCD14 possesses
lectin-like properties and recognizes the inner core of LPS and the
peptidoglycan (9, 12, 17). Within LPS, the major
carbohydrate determinants of the interaction are the KDO sugars
(9), but the N-acylated glucosamine residues of lipid A also
contribute to this recognition (12). It may be hypothesized that the interactions of sCD14 with the sugar moiety of LPS still allow
the recognition of the lipid A by hLf. This hypothesis is supported by
previous data showing that NaCl concentrations above 0.4 M inhibit the
binding of LPS to hLf (47), thus explaining the
saline-labile interactions between hLf and sCD14-LPS.
The evidence for interactions between hLf and sCD14 led us to
investigate whether hLf interferes in the biological activity of the
sCD14-LPS complex. ICAM-1 and E-selectin are adhesion molecules whose
expression is induced by LPS in the presence of sCD14 on endothelial
cells (18, 19, 36). The results show that the expression of
ICAM-1 and E-selectin on HUVEC is strongly induced by O55:B5 E. coli LPS in the presence of sCD14 but that these levels of
expression are inhibited by hLf, whatever the order of presentation of
hLf to sCD14 and LPS. Thus, the binding of hLf to sCD14 in the presence
of LPS leads to complexes, which are then unable to activate
endothelial cells. One could speculate whether the sCD14-hLf complexes
are then still able to bind LPS or not, but in either case, a loss of
the cell-activating properties of LPS will occur. These phenomena could
significantly lower the availability of sCD14 in serum and/or disable
sCD14-LPS complexes, thus further decreasing the responsiveness of the
organism to LPS. In light of these results, it can be assumed that the
septic shock-preventive effect of hLf administered to animals 12 to
24 h before lethal doses of LPS (51) is related, at
least in part, to the ability of hLf to bind sCD14. The delay in the
protective effect of hLf may be a requisite in vivo for the optimal
neutralization of free sCD14 by hLf. It may also be relevant to some
more complex phenomena.
In conclusion, our findings provide evidence that the role of hLf in
the modulation of the inflammatory process cannot be attributed solely
to its LPS-chelating properties (2, 14, 47). We have
previously shown that hLf may compete with LBP for the binding of LPS
to mCD14 (13). We demonstrate here the ability of hLf to
bind to sCD14 and to inhibit at least one of its cell activation
functions, the expression of ICAM-1 and E-selectin, two molecules
essential to the recruitment process of leukocytes. Since CD14 plays an
essential role in the endotoxin-mediated inflammatory response, it is
possible that hLf interferes with the expression and/or activation of
other molecules involved in the leucocyte recruitment process such as
VCAM-1, integrins, and chemokines. The interactions of hLf with CD14
may also account for its previously reported effects on the expression
of proinflammatory cytokines tumor necrosis factor alpha, IL-1, and
IL-6 (10, 29). Therefore, hLf may modulate the recruitment
of immune cells on inflammatory sites and hLf, either released from
neutrophils during inflammation or used as a therapeutic agent, may
have such a modulating effect. These results open the way to
investigate potential interactions between hLf and other LPS receptors
and proinflammatory molecules.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the Ministère de
l'Enseignement et de la Recherche Scientifique, the Centre National de la Recherche Scientifique, the North West Cancer Research Fund, the
Mizutani Foundation for Glycoscience, and the Royal Society. D. G. Fernig was a Université des Sciences et Technologies de Lille-supported visiting professor.
We are grateful to A. Clermont and M. Masson for their skillful
technical assistance. We are indebted to I. Mattsby-Baltzer, who
provided us the different LPS derivatives.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Chimie Biologique, UMR CNRS 8576, Université des Sciences et
Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France. Phone:
33 3 20 33 72 38. Fax: 33 3 20 43 65 55. E-mail:
Dominique.Legrand{at}univ-lille1.fr.
Editor:
R. N. Moore
| |
REFERENCES |
| 1.
|
Anderson, B. F.,
H. M. Baker,
G. E. Norris,
D. W. Rice, and E. N. Baker.
1989.
Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution.
J. Mol. Biol.
209:711-734[CrossRef][Medline].
|
| 2.
|
Appelmelk, B. J.,
Y. Q. An,
M. Geerts,
B. G. Thijs,
H. A. de Boer,
D. M. MacLaren,
J. de Graaff, and J. H. Nuijens.
1994.
Lactoferrin is a lipid A-binding protein.
Infect. Immun.
62:2628-2632[Abstract/Free Full Text].
|
| 3.
|
Arditi, M.,
J. Zhou,
R. Dorio,
G. W. Rong,
S. M. Goyert, and K. S. Kim.
1993.
Endotoxin-mediated endothelial cell injury and activation: role of soluble CD14.
Infect. Immun.
61:3149-3156[Abstract/Free Full Text].
|
| 4.
|
Baveye, S.,
E. Elass,
J. Mazurier,
G. Spik, and D. Legrand.
1999.
Lactoferrin: a multifunctional glycoprotein involved in the modulation of the inflammatory process.
Clin. Chem. Lab. Med.
37:281-286[CrossRef][Medline].
|
| 5.
|
Bazil, V.,
M. Baudys,
I. Hilgert,
I. Stefanova,
M. G. Low,
J. Zbrozek, and V. Horejsi.
1989.
Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14.
Mol. Immunol.
26:657-662[CrossRef][Medline].
|
| 6.
|
Bennett, R. M., and C. Mohla.
1976.
A solid-phase radioimmunoassay for the measurement of lactoferrin in human plasma: variations with age, sex, and disease.
J. Lab. Clin. Med.
88:156-166[Medline].
|
| 7.
|
Bevilacqua, M. P.,
J. S. Pober,
D. L. Mendrick,
R. S. Cotran, and M. A. Gimbrone, Jr.
1987.
Identification of an inducible endothelial-leukocyte adhesion molecule.
Proc. Natl. Acad. Sci. USA
84:9238-9242[Abstract/Free Full Text].
|
| 8.
|
Bone, R. C.
1991.
The pathogenesis of sepsis.
Ann. Intern. Med.
115:457-469.
|
| 9.
|
Cavaillon, J. M.,
C. Marie,
A. Ledur,
I. Godard,
D. Poulain,
C. Fitting, and N. Haeffner-Cavaillon.
1996.
CD14/LPS receptor exhibits lectin-like properties.
J. Endotoxin Res.
3:471-480.
|
| 10.
|
Crouch, S. P.,
K. J. Slater, and J. Fletcher.
1992.
Regulation of cytokine release from mononuclear cells by the iron-binding protein lactoferrin.
Blood
80:235-240[Abstract/Free Full Text].
|
| 11.
|
Dentener, M. A.,
V. Bazil,
E. J. Von Asmuth,
M. Ceska, and W. A. Buurman.
1993.
Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha, IL-6 and IL-8 release by human monocytes and alveolar macrophages.
J. Immunol.
150:2885-2891[Abstract].
|
| 12.
|
Dziarski, R.,
R. I. Tapping, and P. S. Tobias.
1998.
Binding of bacterial peptidoglycan to CD14.
J. Biol. Chem.
273:8680-8690[Abstract/Free Full Text].
|
| 13.
|
Elass-Rochard, E.,
D. Legrand,
V. Salmon,
A. Roseanu,
M. Trif,
P. S. Tobias,
J. Mazurier, and G. Spik.
1998.
Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein.
Infect. Immun.
66:486-491[Abstract/Free Full Text].
|
| 14.
|
Elass-Rochard, E.,
A. Roseanu,
D. Legrand,
M. Trif,
V. Salmon,
C. Motas,
J. Montreuil, and G. Spik.
1995.
Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli O55B5 lipopolysaccharide.
Biochem. J.
312:839-845.
|
| 15.
|
Fernig, D. G.,
P. S. Rudland, and J. A. Smith.
1992.
Rat mammary myoepithelial-like cells in culture possess kinetically distinct low-affinity receptors for fibroblast growth factor that modulate growth stimulatory responses.
Growth Factors
7:27-39[Medline].
|
| 16.
|
Frey, E. A.,
D. S. Miller,
T. G. Jahr,
A. Sundan,
V. Bazil,
T. Espevik,
B. B. Finlay, and S. D. Wright.
1992.
Soluble CD14 participates in the response of cells to lipopolysaccharide.
J. Exp. Med.
176:1665-1671[Abstract/Free Full Text].
|
| 17.
|
Gupta, D.,
T. N. Kirkland,
S. Viriyakosol, and R. Dziarski.
1996.
CD14 is a cell-activating receptor for bacterial peptidoglycan.
J. Biol. Chem.
271:23310-23316[Abstract/Free Full Text].
|
| 18.
|
Hailman, E.,
H. S. Lichenstein,
M. M. Wurfel,
D. S. Miller,
D. A. Johnson,
M. Kelley,
L. A. Busse,
M. M. Zukowski, and S. D. Wright.
1994.
Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.
J. Exp. Med.
179:269-277[Abstract/Free Full Text].
|
| 19.
|
Haziot, A.,
G. W. Rong,
J. Silver, and S. M. Goyert.
1993.
Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide.
J. Immunol.
151:1500-1507[Abstract].
|
| 20.
|
Hess, D. C.,
Y. Thompson,
A. Sprinkle,
J. Carroll, and J. Smith.
1996.
E-selectin expression on human brain microvascular endothelial cells.
Neurosci. Lett.
213:37-40[CrossRef][Medline].
|
| 21.
|
Jaffe, E. A.,
R. L. Nachman,
C. G. Becker, and C. R. Minick.
1973.
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
J. Clin. Investig.
52:2745-2756.
|
| 22.
|
Juan, T. S.,
E. Hailman,
M. J. Kelley,
S. D. Wright, and H. S. Lichenstein.
1995.
Identification of a domain in soluble CD14 essential for lipopolysaccharide (LPS) signaling but not LPS binding.
J. Biol. Chem.
270:17237-17242[Abstract/Free Full Text].
|
| 23.
|
Juan, T. S.,
E. Hailman,
M. J. Kelley,
L. A. Busse,
E. Davy,
C. J. Empig,
L. O. Narhi,
S. D. Wright, and H. S. Lichenstein.
1995.
Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64.
J. Biol. Chem.
270:5219-5224[Abstract/Free Full Text].
|
| 24.
|
Lee, C. H.,
Y. A. Reid,
J. S. Jong, and Y. H. Kang.
1995.
Lipopolysaccharide-induced differential cell surface expression of intercellular adhesion molecule-1 in cultured human umbilical cord vein endothelial cells.
Shock
3:96-101[Medline].
|
| 25.
|
Lee, W. J.,
J. L. Farmer,
M. Hilty, and Y. B. Kim.
1998.
The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets.
Infect. Immun.
66:1421-1426[Abstract/Free Full Text].
|
| 26.
|
Legrand, D.,
J. Mazurier,
P. Maes,
E. Rochard,
J. Montreuil, and G. Spik.
1991.
Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site.
Biochem. J.
276:733-738.
|
| 27.
|
Legrand, D.,
P. H. Van Berkel,
V. Salmon,
H. A. Van Veen,
M. C. Slomianny,
J. H. Nuijens, and G. Spik.
1997.
The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells.
Biochem. J.
327:841-846.
|
| 28.
|
Leveugle, B.,
J. Mazurier,
D. Legrand,
C. Mazurier,
J. Montreuil, and G. Spik.
1993.
Lactotransferrin binding to its platelet receptor inhibits platelet aggregation.
Eur. J. Biochem.
213:1205-1211[Medline].
|
| 29.
|
Machnicki, M.,
M. Zimecki, and T. Zagulski.
1993.
Lactoferrin regulates the release of tumour necrosis factor alpha and interleukin 6 in vivo.
Int. J. Exp. Pathol.
74:433-439[Medline].
|
| 30.
|
Mann, D. M.,
E. Romm, and M. Migliorini.
1994.
Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.
J. Biol. Chem.
269:23661-23667[Abstract/Free Full Text].
|
| 31.
|
Masson, P. L.,
J. F. Heremans, and E. Schonne.
1969.
Lactoferrin, an iron-binding protein in neutrophilic leukocytes.
J. Exp. Med.
130:643-658[Abstract].
|
| 32.
|
Mattsby-Baltzer, I.,
P. Gemski, and C. R. Alving.
1984.
Heterogeneity of lipid A: comparison of lipid A types from different gram-negative bacteria.
J. Bacteriol.
159:900-904[Abstract/Free Full Text].
|
| 33.
|
Mattsby-Baltzer, I.,
A. Roseanu,
C. Motas,
J. Elverfors,
I. Engberg, and L. A. Hanson.
1996.
Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells.
Pediatr. Res.
40:257-262[Medline].
|
| 34.
|
Mayeux, P. R.
1997.
Pathobiology of lipopolysaccharide.
J. Toxicol. Environ. Health
51:415-435[CrossRef][Medline].
|
| 35.
|
Mazurier, J., and G. Spik.
1980.
Comparative study of the iron-binding properties of human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin.
Biochim. Biophys. Acta
629:399-408[Medline].
|
| 36.
|
Pugin, J.,
C. C. Schurer-Maly,
D. Leturcq,
A. Moriarty,
R. J. Ulevitch, and P. S. Tobias.
1993.
Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14.
Proc. Natl. Acad. Sci. USA
90:2744-2748[Abstract/Free Full Text].
|
| 37.
|
Rahmoune, H.,
P. S. Rudland,
J. T. Gallagher, and D. G. Fernig.
1998.
Hepatocyte growth factor/scatter factor has distinct classes of binding site in heparan sulfate from mammary cells.
Biochemistry
37:6003-6008[CrossRef][Medline].
|
| 38.
|
Rahmoune, H.,
H. L. Chen,
J. T. Gallagher,
P. S. Rudland, and D. G. Fernig.
1998.
Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate.
J. Biol. Chem.
273:7303-7310[Abstract/Free Full Text].
|
| 39.
|
Spik, G.,
G. Strecker,
B. Fournet,
S. Bouquelet,
J. Montreuil,
L. Dorland,
H. Van Halbeek, and J. F. Vliegenthart.
1982.
Primary structure of the glycans from human lactotransferrin.
Eur. J. Biochem.
121:413-419[Medline].
|
| 40.
|
Stelter, F.,
H. Loppnow,
R. Menzel,
U. Grunwald,
M. Bernheiden,
R. S. Jack,
A. J. Ulmer, and C. Schutt.
1999.
Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide.
J. Immunol.
163:6035-6044[Abstract/Free Full Text].
|
| 41.
|
Stelter, F.,
M. Pfister,
M. Bernheiden,
R. S. Jack,
P. Bufler,
H. Engelmann, and C. Schutt.
1996.
The myeloid differentiation antigen CD14 is N- and O-glycosylated. Contribution of N-linked glycosylation to different soluble CD14 isoforms.
Eur. J. Biochem.
236:457-464[Medline].
|
| 42.
|
Tapping, R. I., and P. S. Tobias.
1997.
Cellular binding of soluble CD14 requires lipopolysaccharide (LPS) and LPS-binding protein.
J. Biol. Chem.
272:23157-23164[Abstract/Free Full Text].
|
| 43.
|
Tobias, P. S.,
R. I. Tapping, and J. A. Gegner.
1999.
Endotoxin interactions with lipopolysaccharide-responsive cells.
Clin. Infect. Dis.
28:476-481[Medline].
|
| 44.
|
Tobias, P. S.,
K. Soldau, and R. J. Ulevitch.
1989.
Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein.
J. Biol. Chem.
264:10867-10871[Abstract/Free Full Text].
|
| 45.
|
Tobias, P. S.,
J. C. Mathison, and R. J. Ulevitch.
1988.
A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis.
J. Biol. Chem.
263:13479-13481[Abstract/Free Full Text].
|
| 46.
|
Tobias, P. S.,
K. Soldau,
J. A. Gegner,
D. Mintz, and R. J. Ulevitch.
1995.
Lipopolysaccharide binding protein-mediated complexation of lipopolysaccharide with soluble CD14.
J. Biol. Chem.
270:10482-10488[Abstract/Free Full Text].
|
| 47.
|
Van Berkel, P. H.,
M. E. Geerts,
H. A. Van Veen,
M. Mericskay,
H. A. de Boer, and J. H. Nuijens.
1997.
N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.
Biochem. J.
328:145-151.
|
| 48.
|
Viriyakosol, S., and T. N. Kirkland.
1995.
A region of human CD14 required for lipopolysaccharide binding.
J. Biol. Chem.
270:361-368[Abstract/Free Full Text].
|
| 49.
|
Wellicome, S. M.,
M. H. Thornhill,
C. Pitzalis,
D. S. Thomas,
J. S. Lanchbury,
G. S. Panayi, and D. O. Haskard.
1990.
A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide.
J. Immunol.
144:2558-2565[Abstract].
|
| 50.
|
Wright, S. D.,
R. A. Ramos,
P. S. Tobias,
R. J. Ulevitch, and J. C. Mathison.
1990.
CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science
249:1431-1433[Abstract/Free Full Text].
|
| 51.
|
Zagulski, T.,
P. Lipinski,
A. Zagulska,
S. Broniek, and Z. Jarzabek.
1989.
Lactoferrin can protect mice against a lethal dose of Escherichia coli in experimental infection in vivo.
Br. J. Exp. Pathol.
70:697-704[Medline].
|
| 52.
|
Ziegler-Heitbrock, H. W., and R. J. Ulevitch.
1993.
CD14: cell surface receptor and differentiation marker.
Immunol. Today
14:121-125[CrossRef][Medline].
|
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Abstract
Introduction
