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A Synthetic Lipopolysaccharide-Binding
Peptide Based on Amino Acids 27−39 of
Serum Amyloid P Component Inhibits
Lipopolysaccharide-Induced Responses in
Human Blood
Carla J. C. de Haas, Marijke E. van der Tol, Kok P. M. Van
Kessel, Jan Verhoef and Jos A. G. Van Strijp
J Immunol 1998; 161:3607-3615; ;
http://www.jimmunol.org/content/161/7/3607
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Copyright © 1998 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
A Synthetic Lipopolysaccharide-Binding Peptide Based on
Amino Acids 27–39 of Serum Amyloid P Component Inhibits
Lipopolysaccharide-Induced Responses in Human Blood
Carla J. C. de Haas,1 Marijke E. van der Tol, Kok P. M. Van Kessel, Jan Verhoef, and
Jos A. G. Van Strijp
L
ipopolysaccharide is the major component of the outer membrane of Gram-negative bacteria. The specific interaction of
LPS with soluble and cell surface localized components is a
prerequisite for the orchestration of the immune response to a Gramnegative infection. Cell activation requires CD14, which is not only
expressed on monocytes, macrophages, and neutrophils, but is also
present in a soluble form in serum as soluble CD14 (sCD14).2 CD14
is the only known receptor for LPS that is able to transduce a signal,
albeit probably indirectly (1, 2). CD14-negative cells, such as endothelial, epithelial, and smooth muscle cells, can be activated by LPS
via the interaction of LPS with sCD14 (3). LPS-binding protein (LBP)
is a lipid-transfer protein that promotes the movement of LPS from
micelles to (s)CD14 (4). The interaction of LPS with (s)CD14 is
markedly enhanced by LBP and initiates a cascade of cellular responses, which are necessary to fight Gram-negative infection, but
under certain circumstances also lead to septic shock (5). LBP can
also catalyze the movement of LPS to high-density lipoproteins
(HDL), which neutralizes the capacity of LPS to stimulate cells (6, 7).
There are more LPS-binding proteins known to play a role in LPSmediated effects, several of which have been recognized among the
antimicrobial arsenal secreted by neutrophils (8). Bactericidal/permeEijkman Winkler Institute, Department of Inflammation, Utrecht, The Netherlands
Received for publication January 14, 1998. Accepted for publication June 3, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Carla J. C. de Haas, Eijkman
Winkler Institute. Department of Inflammation, AZU, G04.614, Heidelberglaan 100,
3584 CX Utrecht, The Netherlands. E-mail address: [email protected]
2
Abbreviations used in this paper: sCD14, soluble CD14; BPI, bactericidal/permeability-increasing protein; CAP, cationic protein; HDL, high-density lipoprotein;
HSA, human serum albumin; LBP, LPS-binding protein; mCD14, membrane-bound
CD14; ReLPS, LPS from Salmonella minnesota strain R595; SAP, serum amyloid P.
Copyright © 1998 by The American Association of Immunologists
ability-increasing protein (BPI), a cationic protein, not only exerts
strong antimicrobial effects against Gram-negative bacteria, but is also
potent in binding and detoxifying LPS (9, 10). Neutrophil granule
components, such as cationic protein 18 (CAP18), lysozyme, lactoferrin, and azurocidin/CAP37, have also been described as LPS-binding proteins with a neutralizing capacity (8, 11, 12).
Serum amyloid P component (SAP) is a decameric serum glycoprotein composed of identical 25.5-kDa subunits noncovalently
associated in two pentameric rings interacting face to face. It has
been associated with all forms of amyloid deposits, for example
with those in Alzheimer’s and Parkinson’s disease, Down’s syndrome, and Creutzfeldt-Jacob syndrome (13). It is described to
protect amyloid deposits from proteolytic degradation in vivo (14).
Recently, the participation of SAP in the pathogenesis of amyloidosis was demonstrated using mice with targeted deletion of the
SAP gene (15). However, SAP has also been reported to inhibit
Alzheimer b-peptide fibril formation in an in vitro model (16).
Furthermore, it is present in the normal glomerular basement membrane covalently associated with collagen and is associated with
elastic fibers in skin and blood vessels (17). SAP belongs to the
family of pentraxins, lectin-like serum proteins, which have been
stably conserved throughout vertebrate evolution. This protein has
a 51% amino acid homology with C-reactive protein, the classical
acute-phase protein found in humans. SAP is an acute-phase reactant in mice, while it is constitutively present in human serum at
40 mg/ml, with a maximum twofold increase during sepsis (14).
SAP shows calcium-dependent binding to DNA (18), chromatin
(19), and glycosaminoglycans such as heparin, heparan, and dermatan sulfate (20), and has been described to play a role in the
complement cascade since it can bind to several complement components, such as C4b-binding protein, C1q, and C3bi (21, 22), and
to immune complexes, probably via the F(ab9)2 fragment of IgG
0022-1767/98/$02.00
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LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been
attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins,
leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic
beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component
(SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled
ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst
of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that
pep27–39, a 13-mer peptide consisting of amino acids 27–39 of SAP, competitively inhibited the binding of LPS to SAP. In addition,
pep27–39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole
blood. Carboxamidomethylated pep27–39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could
not prevent binding of LPS to soluble CD14, in contrast to pep27–39. The ability of pep27–39 to antagonize specifically the effects
of LPS in the complex environment of human blood suggests that pep27–39 may be a novel therapeutic agent in the treatment of
Gram-negative sepsis. The Journal of Immunology, 1998, 161: 3607–3615.
3608
SAP PEPTIDE INHIBITS LPS RESPONSES IN HUMAN BLOOD
(23, 24). Although its exact physiologic function is still unknown,
it is believed to play a role in the binding and clearance of host- or
pathogen-derived cellular debris at sites of inflammation (25).
At present, it is still not possible to reconstruct the actual modulating role of plasma in phagocyte activation by LPS and the
pathophysiology of Gram-negative sepsis. Therefore, we sought
other components that fulfill an important role in these events.
creasing amounts (1–100 mg/ml) of lipid A, the rough types (Re, Rc, Ra)
of LPS from S. typhimurium, LPS from S. minnesota R595, LPS from E.
coli O111:B4, or from its Rc-mutant J5; washed three times with HBSS
containing 0.05% Tween-20; and incubated with mouse anti-human SAP
mAb clone 5 (Sigma) for 30 min at room temperature with a subsequent
washing procedure. Detection of SAP binding to the beads was performed
by incubating the beads for 30 min with FITC-labeled goat anti-mouse Ig
(Becton Dickinson, Mountain View, CA) and analysis on a FACScan (Becton Dickinson).
Materials and Methods
Isolation of SAP from serum
Reagents
LPS from Salmonella minnesota strain R595, Escherichia coli O111:B4,
its Rc mutant J5, monophosphoryl lipid A, and LPS from Salmonella typhimurium and S. typhimurium TV119 (Ra), SL684 (Rc), and SL1181 (Re)
were obtained from Sigma (St. Louis, MO). Human rLBP was a generous
gift from H. Lichenstein (Amgen, Boulder, CO).
Serum and plasma
Peptide synthesis
A 13-mer peptide, pep27–39 (EKPLQNFTLCFRA), corresponding to
amino acids 27–39 of SAP, and a scrambled peptide, pep27–39scr
(TRLAFPKECLNQF), were prepared by automated simultaneous multiple
peptide synthesis. The simultaneous multiple peptide synthesis setup was
developed using a standard autosampler, as described previously (26).
Briefly, standard Fmoc chemistry with in situ PyBop/N-methylmorpholine
(NMM) (Novabiochem, Laufelfingen, Switzerland) activation of the amino
acids in a fivefold molar excess with respect to 2 mmol/peptide PALPEG-PS resin (Perseptive Biosystems, Framingham, MA) was employed.
Peptides were obtained as C-terminal amides after cleavage with 90 to 95%
trifluoroacetic acid/scavenger mixtures. Peptides were dissolved in 50 mM
HAc at a concentration of 5 mM and further diluted in 0.25 M Tris-HCl,
pH 7.5, to a concentration of 0.6 mM. Before use in biologic assays, the
peptides were further diluted in HBSS, containing 0.2% human serum albumin (HSA; Central Laboratory Blood transfusion, Amsterdam, The
Netherlands). In some experiments, pep27–39 was carboxamidomethylated
to prevent formation of dimers, as follows: Pep27–39 in 50 mM HAc was
first reduced using Tris(2-carboxyethyl)phosphine hydrochloride (TCEPHCl; Pierce, Rockford, IL; 2 mg/1.5 mmol peptide) for 1.5 h at room
temperature under constant agitation. Then the pH was adjusted to pH 7
using 4 M NaOH, and 100 ml 0.5 M iodoacetamide (Merck-Schuchardt,
Hohenbrunn bei Munchen, Germany) was added. Carboxamidomethylation was allowed to take place overnight under constant agitation. Carboxamidomethylated pep27–39 was then dialyzed against 50 mM HAc in
a 100 D cutoff dialysis membrane. Ellman’s reagent (5, 59-dithio-bis-(2nitrobenzoic acid); Sigma) (27) was used to determine the presence of
free sulfhydryl groups before and after carboxamidomethylation. Optimal
reduction was verified and subsequent efficient carboxamidomethylation
was confirmed by the total absence of free sulfhydryl groups after the
procedure.
Detection of LPS-binding proteins in serum and plasma
LPS from S. minnesota strain R595 (ReLPS) was used to coat magnetic
beads (Tosyl-activated Dyna M-450 beads; Dynal A. S., Oslo, Norway), as
described by Weersink et al. (28). Control beads were treated similarly
without the addition of ReLPS. For the detection of LPS-binding proteins,
ReLPS-coated magnetic beads (5 3 106) were incubated with 300 ml 10%
human serum, heparin plasma, or EDTA plasma for 30 min at 37°C on a
rotator. Except for EDTA plasma, which was diluted in PBS, samples were
diluted in HBSS. After incubation, the beads were washed three times with
HBSS containing 0.05% Tween-20. Proteins were eluted from the beads by
heating at 100°C for 2 min in 20 ml sample buffer (2% SDS, 2.5% DTT,
20% glycerol, 0.001% bromophenol blue, in 0.05 M Tris-HCl, pH 6.9) and
detected by SDS-PAGE on 12.5%, 0.75 mm minigels. Gels were stained
with Coomassie brilliant blue. Proteins were blotted onto Problott membrane, stained with Coomassie brilliant blue, according to the manufacturer’s descriptions (Applied Biosystems, Foster City, CA), and sequenced on
an Applied Biosystems protein sequencer model 476A. In other experiments, magnetic beads were coated with 1 mg/ml smooth type LPS from
S. typhimurium. The beads (5 3 106) were then incubated with 1 mg/ml
SAP for 30 min at 37°C, under constant agitation, in the presence of in-
Cell isolation
Human neutrophils and PBMC were isolated from heparinized blood
drawn from healthy volunteers, as described by Troelstra et al. (30).
Binding of fluorescein-labeled ReLPS to human monocytes
FITC-labeled ReLPS (FITC-LPS) was prepared as described by Troelstra
et al. (31), with a molar labeling efficiency of 1:1. For FITC-LPS binding
studies, 2.5 ng/ml FITC-LPS was preincubated with increasing amounts of
SAP (0 –30 mg/ml) or pep27–39 (0 –10 mM) for 0 to 30 min at 37°C in
HBBS containing 0.2% HSA. Then LBP (10 ng/ml) and PBMC (6 3
106/ml) in the same buffer were added to a final volume of 50 ml, gently
shaken for 30 min at 37°C, and put on ice. Binding of FITC-LPS to monocytes was analyzed on a FACScan, using forward and sideward scatter
parameters to gate on monocytes. The results were expressed as the mean
fluorescence of 10,000 cells. The percentage of inhibition of binding was
calculated using the following formula: 1-(A-bgrA/B-bgrB) 3 100%,
where A is the mean fluorescence of cells incubated with FITC-LPS 1
LBP 1 SAP or pep27–39; B, the mean fluorescence of cells incubated with
FITC-LPS 1 LBP; bgrA, the background fluorescence of cells incubated
with FITC-LPS 1 SAP or pep27–39; and bgrB, the background fluorescence of cells incubated with FITC-LPS alone.
LPS-induced priming of human neutrophils
This procedure has been described in detail elsewhere (30). Briefly, neutrophils (5 3 106/ml) were added to a mixture of 1 ng/ml LPS alone or 1
ng/ml LPS with increasing amounts of SAP (0 –30 mg/ml) or peptides
(0 –30 mM) in the presence of 1 to 100 ng/ml LBP in HBBS/1.8% HSA. In
some experiments, increasing amounts of serum (0.1–3%) were used. In
experiments using peptides, LPS and peptides were preincubated for 30
min before addition to the cells. Cells were incubated with the mixtures for
30 min at 37°C under constant agitation. Next, chemoluminescence response was measured in a luminometer (Autolumat LB 953; Berthold
GmbH, Wildbad, Germany) after automated injection of FMLP (1 mM
final concentration) and HBSS containing 180 mM luminol (Sigma). The
chemoluminescence response was measured automatically over a period of
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Blood was drawn from healthy human volunteers. Human serum was obtained after pooling the sera of three or more donors and stored until use at
270°C. Plasma was obtained from blood in heparinized or EDTA tubes.
Fresh human serum was used for SAP isolation.
Isolation of SAP from serum was performed as described by Skinner and
Cohen (29) with modifications. Briefly, fresh human serum was centrifuged at 17,000 3 g for 5 h at 4°C to remove the top lipid layer. The
delipidated serum was then applied to a Sepharose 4B (Pharmacia, Uppsala, Sweden) column, equilibrated with a calcium buffer (140 mM NaCl,
0.01 M Tris-HCL, 2 mM CaCl2, pH 7.8). SAP was eluted with an EDTA
buffer (140 mM NaCl, 0.01 M Tris-HCl, 10 mM EDTA, pH 8) and applied
to a gel-filtration column (Superdex 200; Pharmacia) equilibrated in the
same EDTA buffer. Fractions containing SAP were concentrated in an
Amicon filter system (10-kDa cutoff) and dialyzed against PBS or saline.
Purity of the SAP isolate was checked with SDS-PAGE, and subsequent
Coomassie brilliant blue staining. The SAP concentration was determined
by ELISA. Therefore, microtiter plates (96-well polyvinyl; Costar, Cambridge, MA) were coated overnight at 4°C with anti-human SAP mAb 5.4A
in PBS (1 mg/ml; Monosan; Sanbio, Uden, The Netherlands) and blocked
for 1 h at 37°C with PBS/0.05% Tween/4% BSA. Samples and a SAP
standard (Calbiochem-Novabiochem, La Jolla, CA) were diluted in PBS/
Tween/1% BSA and incubated for 1 h at 37°C, followed by a 1-h incubation with a second biotinylated anti-human SAP mAb 5.4A (1 mg/ml;
Monosan; Sanbio). Then peroxidase-labeled streptavidin (Southern Biotechnology, Birmingham, AL) was added, and after 1 h, the substrate composed of TMB (tetramethylbenzidine; Sigma) and H2O2 in 0.1 M acetate
buffer was allowed to be converted for 10 min. To stop the enzymatic
reaction, 2 N H2SO4 was added and the OD was determined using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA) operating at 450
nm. In between incubations, the plate was washed five times with H2O/
0.05% Tween. LPS contamination of SAP preparations was about 10 to 20
ng/mg SAP, as determined by the Limulus amebocyte lysate assay (Chromogenix AB, Mölndal, Sweden).
The Journal of Immunology
3609
10 min. Data were analyzed with the AXIS software package (ExOxEmis,
San Antonio, TX). Curves were obtained for all samples presenting the
chemoluminescence response in cpm versus time. Absolute counts were
obtained by calculating the area under the curve of the chemoluminescence
for 10 min. In experiments using human blood, 80 ml of human blood was
incubated with 20 ml of LPS/peptide mixture for 30 min at 37°C. Then 900
ml PBS/0.05% glucose was added, and 100 ml of this mixture was used to
measure the chemoluminescence response, as described. In some experiments, PMA (25 ng/ml) was used to activate the neutrophils or blood for
a chemoluminescence response. In other experiments, TNF-a (1 nM) was
used to prime neutrophils or blood for an enhanced FMLP response in the
presence of peptides.
J5-LPS ELISA
FIGURE 1. SDS-PAGE of proteins extracted from serum and plasma
using ReLPS-coated beads. ReLPS-coated beads (A) and control beads (B)
were incubated with 10% serum (lane 1), EDTA plasma (lane 2), and
heparin plasma (lane 3) for 30 min. Beads were washed and heated for 3
min at 100°C in sample buffer. Eluted proteins were detected on SDSPAGE with Coomassie brilliant blue staining. M represents the molecular
mass (kDa) of marker proteins.
FITC-LPS gel filtration
To study the effect of SAP and pep27–39 on the LPS binding to recombinant sCD14 (rsCD14; kindly provided by Dr. Henri S. Lichenstein, Amgen), gel filtration of FITC-LPS in combination with on-line fluorescence
detection was used. In principle, this system resembles the gel-shift assay
described earlier by Hailman et al. (32) using 3H-labeled LPS. FITC-LPS
alone forms self-quenching aggregates. This LPS aggregate will migrate as
a molecule of about 500 kDa with a very low fluorescence signal. Addition
of sCD14 will monomerize FITC-LPS, resulting in a rise in fluorescence of
the rsCD14/FITC-LPS complex and a comigration of FITC-LPS with
rsCD14. FITC-LPS (0.5 mg/ml) was incubated with 5 mg/ml rsCD14 and
100 ng/ml LBP with or without addition of 100 mg/ml SAP or 10 to 30 mM
of pep27–39. Pep27–39 was preincubated with FITC-LPS for 30 min at
37°C before addition of rsCD14 and LBP. After 30-min incubation at
37°C, 100 ml of the mixture was loaded onto a Superdex TM 200 HR 10/30
column (Pharmacia) and run at a flow of 0.5 ml/min for 35 ml. The effect
of SAP and pep27–39 on the binding of LPS to rsCD14 was also studied
in the absence of LBP. In these experiments, the incubation time of rsCD14
and FITC-LPS was also 30 min. To determine the retention time of FITCLPS, a sample of 50 mg/ml FITC-LPS alone was run. Fluorescence was
recorded using a Perkin-Elmer (Norwalk, CT) LS30 luminometer with excitation wavelength of 475 nm and emission set at 514 nm.
These data suggest that SAP has a specific binding capacity for
LPS. As shown in Figure 3, experiments performed with beads
coated with smooth type LPS from S. typhimurium demonstrated
that purified SAP can also bind to a smooth type of LPS. Competition experiments with other rough and smooth types of LPS from
E. coli and other Salmonella strains showed that SAP not only
exhibits a specific binding to ReLPS from S. minnesota, but it also
specifically binds to all other tested forms of LPS, including monophosphoryl lipid A.
SAP inhibits binding of FITC-LPS to human monocytes
The finding that SAP specifically binds to LPS prompted us to
investigate the effect of SAP on the LPS-induced effects on phagocytes. For this purpose, we studied the effect of purified SAP on
LPS binding to monocytes. FITC-LPS was preincubated with various amounts of SAP, whereafter the binding of FITC-LPS to
monocytes was studied by flow cytometry. As shown in Figure 4,
Results
Detection of LPS-binding proteins in human serum and plasma
Magnetic beads coated with LPS from S. minnesota strain R595
(ReLPS beads) were used to capture LPS-binding proteins from
serum and plasma samples. Figure 1A shows the binding of three
proteins, with Mr values of approximately 70, 45, and 30 kDa, in
the presence of 10% serum and heparin plasma, while hardly any
proteins bound to the ReLPS beads in the presence of 10% EDTA
plasma. Control nonReLPS-coated beads showed binding of the
70-kDa protein only (Fig. 1B). N-terminal amino acid sequencing
of the blotted 45- and 70-kDa proteins showed 100% homology
with the a- and the b-chain of complement component C3bi, respectively. The 30-kDa protein yielded a sequence of 17 amino
acids that was 100% homologous with that of the N-terminal sequence of human SAP. To check whether binding of these proteins
to ReLPS was specific, we incubated ReLPS beads in 10% serum
in the presence of increasing amounts of free ReLPS. Figure 2
shows that in the presence of 10 or 100 mg/ml free ReLPS, binding
of the 30-kDa protein to the beads was inhibited, suggesting competition between free LPS and LPS coated on the beads for binding
to SAP. Additional experiments showed that purified SAP, isolated
from human serum, also bound to ReLPS beads. This binding
could be inhibited by the addition of free ReLPS (data not shown).
FIGURE 2. SDS-PAGE showing binding of SAP to ReLPS-coated
beads and to free ReLPS. ReLPS-coated beads were incubated with 10%
serum for 30 min, then washed and heated for 3 min at 100°C in sample
buffer. Eluted proteins were detected on SDS-PAGE with Coomassie brilliant blue staining. Lanes 1 to 4 show proteins binding to ReLPS-coated
beads in the absence (lane 1) and presence of 100 mg/ml (lane 2), 10 mg/ml
(lane 3), and 1 mg/ml (lane 4) free ReLPS. M represents the molecular
mass (kDa) of marker proteins.
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J5-LPS was coated to 96-well flat-bottom plates (Greiner, Nürtingen, Germany) at a concentration of 1 mg/ml in PBS for 1 h at 37°C, with a subsequent overnight incubation at 4°C. The plate was washed five times with
H2O/0.05% Tween, and blocked for 1 h at 37°C with PBS/4% BSA/0.05%
Tween. Then 0.3 mg/ml SAP was incubated with increasing concentrations
of pep27–39 (0 –30 mM) in HBSS/0.2% BSA/0.05% Tween for 1 h at
37°C. Subsequently, the binding of SAP was detected, as described for the
SAP ELISA in Materials and Methods (isolation of SAP from serum), with
the only exception of using HBSS/0.2% BSA/0.05% Tween as a dilution
buffer for the second biotinylated anti-human SAP 5.4A mAb and the peroxidase-labeled streptavidin.
3610
SAP PEPTIDE INHIBITS LPS RESPONSES IN HUMAN BLOOD
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FIGURE 3. Flow-cytometric analysis of SAP binding to various LPS
types and monophosphoryl lipid A. Magnetic beads were coated with
smooth type LPS from S. typhimurium. Binding of 1 mg/ml SAP to the
beads was tested in the presence of increasing amounts (1–100 mg/ml) of
monophosphoryl lipid A, smooth and rough types of LPS from S. typhimurium, LPS from S. minnesota R595, and LPS from E. coli O111:B4 and
its Rc mutant J5. SAP binding was determined by incubating the beads
with an anti-human SAP mAb and then FITC-labeled goat anti-mouse Ig.
Beads were analyzed on a FACScan. Binding of SAP to the beads without
the addition of one of the aforementioned free types of LPS represents
100% SAP binding. Data represent the mean of two separate experiments.
preincubation of FITC-LPS with SAP dose dependently inhibited
the binding of FITC-LPS to monocytes up to 90%. This effect was
already evident at a concentration of 1 mg/ml SAP. To study kinetics of SAP binding to LPS, FITC-LPS was preincubated for 0
to 30 min with 10 mg/ml SAP, and the competence of FITC-LPS
to bind to monocytes was analyzed. Even without preincubation,
SAP inhibited binding of FITC-LPS to monocytes to the same
extent as was achieved after 30 min of preincubation (data not
shown). To exclude the possibility that SAP prevents LPS binding
to the monocytes, via binding to the monocytes itself, a control
experiment was performed. Therefore, PBMC were preincubated
with SAP for 30 min, and washed three times to remove unbound
SAP. Subsequent incubation of the PMBC with FITC-LPS, and
analysis on the FACS, showed that preincubation of PBMC with
SAP did not inhibit binding of FITC-LPS to monocytes.
SAP inhibits LPS-induced priming of human neutrophils
To evaluate the effects of SAP on LPS toxicity in a functional
assay, we investigated whether SAP could inhibit LPS-induced
priming of neutrophils. Neutrophils were primed with 1 ng/ml LPS
for an enhanced FMLP response in the presence of 1 ng/ml LBP
for 30 min. Addition of SAP revealed a dose-dependent inhibition
on LPS-induced priming of neutrophils with a 70 to 80% inhibition
at 30 mg/ml SAP (Fig. 5). However, the inhibitory effect of SAP on
LPS-induced priming of neutrophils was profoundly reduced when
higher concentrations of LBP were used (Fig. 6). In the presence
of serum concentrations over 0.1%, the inhibitory effect of SAP on
LPS-induced priming was strongly reduced as well (data not
shown).
SAP peptide, pep27–39, inhibits binding of SAP to LPS
In literature, a peptide, comprising the amino acids 27–39 of the
SAP sequence, was described to interfere with the binding of SAP
FIGURE 4. Flow-cytometric analysis demonstrating inhibition of
FITC-LPS binding to monocytes by SAP. Human monocytes were incubated with FITC-LPS (2.5 ng/ml) in the presence or absence of increasing
amounts of SAP for 30 min. FITC-LPS binding was assessed by FACScan
analysis. FITC-LPS binding to monocytes is shown in the presence of 10
ng/ml LBP without SAP (A), and with 1 (B), 3 (C), 10 (D), and 30 (E)
mg/ml SAP. The background binding of FITC-LPS in the absence of LBP
is shown in all figures (thin lines). The data are representative of five
separate experiments. Percentage of inhibition of FITC-LPS binding to
monocytes is shown in the presence of increasing amounts of SAP 6 SEM
(n 5 5) (F).
The Journal of Immunology
3611
FIGURE 5. Chemoluminescence assay illustrating inhibition of LPSinduced priming of the oxidative burst in human neutrophils by SAP. Human neutrophils were incubated with 1 ng/ml ReLPS in the presence or
absence of increasing amounts of SAP for 30 min, after which the FMLPinduced chemoluminescence was measured. Effects of SAP on LPS priming of neutrophils are shown with LPS together with increasing amounts of
SAP (0 –30 mg/ml), all in the presence of 1 ng/ml LBP. Background luminescence was measured with 1 ng/ml LPS without addition of LBP. The
data are representative of four separate experiments (A). Percentage of
inhibition of the 10-min integral (AUC) of the LPS-induced priming of
neutrophils is shown in the presence of increasing amounts of SAP 6 SEM
(n 5 4) (B).
interfere with the binding of FITC-LPS to monocytes (Fig. 8). A
control experiment testing the possibility that binding of pep27–39
to the monocytes would influence subsequent binding of FITCLPS, as we performed earlier for SAP, showed that prebound
pep27–39 did not influence the binding of FITC-LPS to the monocytes. Testing the effect of pep27–39 on the LPS-induced priming
of neutrophils in the presence of increasing concentrations of LBP,
it was found that 3 mM of pep27–39 profoundly reduced the LPSinduced priming of neutrophils, even at high concentrations of
LBP (Fig. 9). Also in the presence of serum, 30 mM of pep27–39
was able to almost completely inhibit the LPS-induced priming of
neutrophils (1% serum; percentage of inhibition, 90.2 6 11.7;
to some of its ligands. Therefore, we tested whether this peptide
(pep27–39) could interfere with the binding of SAP to LPS as well.
A microtiterplate was coated with J5-LPS and incubated with SAP
in the presence of increasing amounts of pep27–39. Figure 7 shows
that the binding of SAP to J5-LPS was completely inhibited by 30
mM of pep27–39. A control peptide, comprising the same amino
acids in a scrambled order (pep27–39scr), did not interfere with
SAP binding to LPS.
Pep27–39 neutralizes LPS in human serum and blood
Pep27–39 was anticipated to inhibit binding of FITC-LPS to
monocytes. In a flow-cytometric assay, as little as 0.1 mM of
pep27–39 inhibited the binding of FITC-LPS to monocytes by
50%, while a complete inhibition was reached at a concentration of
3 mM of pep27–39. The scrambled peptide, pep27–39scr, did not
FIGURE 7. Pep27–39 inhibits binding of SAP to J5-LPS. SAP binding
to J5-LPS, coated to a 96-well plate, was monitored in the presence of
increasing amounts of pep27–39 or scrambled pep27–39 (pep27–39scr) via
detection with a biotinylated anti-human SAP mAb, followed by a streptavidin-horseradish peroxidase conjugate. Data represent three separate experiments 6 SEM.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 6. LBP decreases the SAP inhibition of the LPS-induced priming of the oxidative burst in human neutrophils. Neutrophils were primed
for 30 min with 1 ng/ml ReLPS and 30 mg/ml SAP in the presence of
increasing amounts of LBP. Thereafter, FMLP-induced chemoluminescence was measured for 10 min. Data represent the percentage of inhibition
of the 10-min integral (AUC) of four separate experiments 6 SEM.
3612
n 5 3). As pep27–39 contains a Cys residue, it will spontaneously
form dimers. To investigate the effect of dimerization of pep27–39
on its LPS-inhibitory effects, the free sulfhydryl group of the Cys
residue was blocked by carboxamidomethylation. In human whole
blood, 30 mM of pep27–39 decreased the LPS-induced priming of
neutrophils to about 50% compared with the chemoluminescence
response in the presence of 1 ng/ml LPS alone. Carboxamidomethylated pep27–39 was even more efficient in inhibiting the
LPS-induced priming of human blood (Fig. 10). The peptide with
the scrambled sequence, pep27–39scr, showed no effect. In addition, carboxamidomethylated pep27–39 was more potent than
pep27–39 in the inhibition of FITC-LPS binding to monocytes
(IC50 of 0.03 mM compared with 0.1 mM). Control experiments
FIGURE 9. Pep27–39 inhibits the LPS-induced priming of neutrophils
independent of the LBP concentration. Human neutrophils were primed for
30 min with 1 ng/ml ReLPS and 3 mM pep27–39 in the presence of increasing amounts of LBP. Subsequently, FMLP-induced chemoluminescence was measured for 10 min. Data represent the percentage of inhibition
of the 10-min integral (AUC) of five separate experiments 6 SEM.
FIGURE 10. Pep27–39 and carboxamidomethylated pep27–39 inhibit
LPS priming of human blood. Human blood was primed for 30 min with
1 ng/ml ReLPS and increasing amounts of peptides. An FMLP-induced
chemoluminescence in 10-fold diluted blood was measured for 10 min.
Data represent the percentage of inhibition of the 10-min integral (AUC) of
four separate experiments 6 SEM.
using TNF-a as the primer for the FMLP-induced oxidative burst
of human blood did not show any inhibitory effects of pep27–39 or
carboxamidomethylated pep27–39. The activation of human blood
by PMA was not affected by either of the peptides (data not
shown). This indicates that (carboxamidomethylated) pep27–39
specifically antagonizes the LPS-induced priming of neutrophils
and that this inhibitory effect was not caused by nonspecific
cytotoxicity.
SAP-derived peptide, pep27–39, but not SAP, inhibits binding of
LPS to rsCD14
To study the effect of SAP and pep27–39 on the LPS binding to
sCD14, we used a FITC-LPS gel-filtration technique in which we
determined the capacity of rsCD14 to bind FITC-LPS, by monitoring the change in retention time of FITC-LPS on a gel-filtration
column, in the presence of SAP or pep27–39. In Figure 11, we
show that FITC-LPS runs at about 7 ml, just after the void volume
of this column, representing 500 kDa (Fig. 11A). A 30-min preincubation of FITC-LPS with rsCD14 and LBP resulted in a shift
of fluorescence from this quenched fluorescence signal at 7 to 14
ml (60 kDa), the place at which rsCD14 elutes from the column
(Fig. 11B). Addition of SAP did not decrease the 60-kDa signal
(data not shown). Even without LBP, SAP could not inhibit binding of FITC-LPS to rsCD14 (Fig. 11F). Lower concentrations of
rsCD14 did not result in a retention time shift, so that the effect of
SAP could not be tested at these concentrations of rsCD14. However, preincubation of FITC-LPS with 30 mM of pep27–39 very
potently inhibited the binding of FITC-LPS to rsCD14, as is shown
by the disappearance of the 60-kDa peak (Fig. 11D). The concentrations of FITC-LPS in Figure 11, B to G, are 10-fold lower then
in Figure 11A. Therefore, the 500-kDa peak, representing the
quenched form of FITC-LPS aggregates, is not visible in the lower
panels. Figure 11, E and G, show that pep27–39 also prevents
binding of LPS to rsCD14 in the absence of LBP.
Discussion
In literature, SAP is described to bind many ligands, although no
clear biologic function has been ascribed to it as yet (19, 20, 22,
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 8. Pep27–39 inhibits binding of FITC-LPS to human monocytes. Human monocytes were incubated with 2.5 ng/ml FITC-LPS and 10
ng/ml LBP for 30 min in the presence of increasing amounts of pep27–39
or scrambled pep27–39 (pep27–39scr). Binding of FITC-LPS was analyzed on a FACScan. Data represent the percentage of inhibition of FITCLPS binding of five separate experiments 6 SEM.
SAP PEPTIDE INHIBITS LPS RESPONSES IN HUMAN BLOOD
The Journal of Immunology
3613
33, 34). In our study, using magnetic beads coated with ReLPS, we
identified SAP as a new LPS-binding protein present in human
plasma. Competition experiments showed that SAP specifically
binds to rough as well as smooth types of LPS; however, the affinity of SAP for smooth types of LPS seems less as compared
with rough types of LPS (Fig. 4). This can be partly explained by
differences in m.w. of the LPS, but the affinity of SAP to smooth
types of LPS could also be lower because of sterical hindrance
caused by the oligosaccharide chain of smooth LPS. Monophosphoryl lipid A was a very potent inhibitor for the binding of SAP
to LPS, indicating that SAP binds to the lipid A part of LPS.
We showed that SAP profoundly inhibited LPS responses in
human granulocytes in the presence of low concentrations of LBP.
This interference of LPS binding to CD14 by SAP was not the
result of direct SAP binding to CD14, thereby inhibiting LPS/
CD14 interactions, as preincubation of monocytes with SAP did
not affect subsequent binding of LPS to the cells (data not shown).
SAP was not able to neutralize LPS in serum or human blood.
However, we demonstrated that SAP binds to LPS in the presence
of serum, suggesting a role for SAP binding to LPS in vivo. Not
much is known about the fate of LPS in vivo. To date, LPS has
been described to bind either (s)CD14 or HDL, after entering the
circulation, via interaction with LBP (4, 6). In the circulation, LPSbinding proteins as BPI and CAP18 cannot play a role in the immediate binding of LPS since they are constituents of neutrophil
granules and are supposed to play a role in LPS neutralization only
at specific sites of inflammation (8, 10, 35). Because the binding of
LPS to HDL is a slow process (6, 36) and we have shown that
binding of SAP to LPS occurs rapidly, we propose that, on entering the circulation, LPS is immediately captured by SAP. Although SAP is not able to neutralize LPS in vivo directly, it could
serve as a carrier protein to transport LPS to the liver for rapid
detoxification, and thus indirectly contribute to LPS clearance. Experiments in SAP-knockout mice are needed to further investigate
the exact role of SAP binding to LPS in endotoxemia.
It has been shown that SAP can interact with phagocytes (37,
38). It can prime neutrophils (37) and enhance macrophage listericidal activity (39, 40). Furthermore, it has been described that
substrate-bound SAP can activate C3b and C3bi receptors of
monocytes (38). Since SAP can bind to phagocytes, bacteria (41,
42), and complement components (21–24), it might serve a role as
an opsonin, potentiating phagocytosis of C3- or SAP-coated pathogens. The direct interaction with LPS on bacteria clearly fits in this
model.
In search for the LPS-binding region of SAP, we found a SAP
peptide, pep27–39, which could compete for the binding of SAP to
LPS. This 13-mer synthetic SAP peptide, comprising the amino acids
27–39 of SAP, was described to interfere with the interaction of SAP
with heparin and C4b-binding protein (21, 33, 43). In addition, a 12mer synthetic peptide that corresponds to amino acids 27–38 was
reported to support cell attachment (44). We showed that pep27–39
was able to inhibit LPS responses in human phagocytes even in the
presence of human blood. Carboxaminomethylation of pep27–39,
which prevents formation of dimers via blockage of the free sulfydryl
groups of Cys residues, resulted in a peptide that was about 4 times
more active. In other studies concerning cell attachment, it was shown
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FIGURE 11. SAP cannot prevent binding of LPS to sCD14, in contrast to pep27–39. FITC-LPS (0.5 mg/ml), incubated with 5 mg/ml sCD14, in the
presence (B, C, D) or absence (E, F, G) of 100 ng/ml LBP, was run on a gel-filtration column in combination with on-line fluorescence detection. Line
A shows the retention time of aggregated FITC-LPS (50 mg/ml) in the absence of sCD14. FITC-LPS was preincubated with 10 mM (C) and 30 mM (D
and G) pep27–39 or with 100 mg/ml SAP (F). The y-axis represents relative fluorescence units. The different trails are superimposed with the same
fluorescence scale for each line.
3614
SAP PEPTIDE INHIBITS LPS RESPONSES IN HUMAN BLOOD
FIGURE 12. Comparison of the LPS-binding motifs of several LPS-binding proteins with the proposed LPS-binding region of SAP. Basic residues are
marked in boldface. Hydrophobic residues are italicized.
peptide pep27–39 is capable of preventing LPS binding to mCD14
as well as sCD14.
We discovered SAP as a novel LPS-binding protein in human
plasma. As SAP did not neutralize LPS responses in human blood,
its role in the pathophysiology of Gram-negative infections has yet
to be elucidated. However, a 13-mer peptide, pep27–39, derived
from SAP was found to bind to LPS. Its carboxamidomethylated
form was even more potent in binding to LPS. The ability of
pep27–39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27–39 may be
a novel therapeutic agent in the defense against Gram-negative
sepsis.
We are currently investigating the capacity of other SAP-derived peptides to bind and neutralize LPS.
Acknowledgments
We thank Fridolin van der Lecq and Dr. Ton Aarsman from the Sequence
Center Utrecht (Institute of Biomembranes, Utrecht University, The Netherlands) for their help in obtaining protein sequences, and Dr. Ruurd van
der Zee (Institute of Infectious Diseases and Immunology, Utrecht University) for the synthesis of the peptides.
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that 83% of the initial activity of the SAP peptide, pep27–38, was
confined to a hexapeptide, pep33–38 (44). When heparin binding was
studied, pep33–38 was even found to have 10-fold higher activity
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The LPS-binding motifs of several LPS-binding proteins have
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for binding to LPS. Another explanation might be the fact that SAP
is a rather large protein. Therefore, pep27–39, only 13 amino acids
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