Previous Article | Next Article 
Infection and Immunity, March 1999, p. 1353-1358, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Neutralization of Endotoxin In Vitro and In Vivo by
a Human Lactoferrin-Derived Peptide
Gui-Hang
Zhang,1,*
David M.
Mann,2,3 and
Chao-Ming
Tsai1
Division of Bacterial Products, Center for
Biologics Evaluation and Research, Food and Drug Administration,
Rockville, Maryland 208521; J. H. Holland Laboratory, Plasma Derivatives Department, American Red
Cross, Rockville, Maryland 208552; and
Department of Biochemistry and Molecular Biology and the
Institute for Biochemical Sciences, George Washington University
Medical Center, Washington, D.C. 200373
Received 31 July 1998/Returned for modification 28 September
1998/Accepted 11 December 1998
 |
ABSTRACT |
Endotoxin (lipopolysaccharide [LPS]) is the major pathogenic
factor of gram-negative septic shock, and endotoxin-induced
death is associated with the host overproduction of tumor necrosis
factor alpha (TNF-
). In the search for new antiendotoxin molecules, we studied the endotoxin-neutralizing capacity of a human
lactoferrin-derived 33-mer synthetic peptide
(GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP; designated LF-33) representing the
minimal sequence for lactoferrin binding to glycosaminoglycans. LF-33
inhibited the coagulation of the Limulus amebocyte
lysate and the secretion of TNF- by RAW 264.7 cells induced by
lipid A and four different endotoxins with a potency
comparable to that of polymyxin B. The first six residues at the N
terminus of LF-33 were critical for its antiendotoxin activity. The
endotoxin-neutralizing capacity of LF-33 and polymyxin B was attenuated
by human serum. Coinjection of Escherichia coli LPS (125 ng) with LF-33 (2.5 µg) dramatically reduced the lethality of LPS in
the galactosamine-sensitized mouse model. Significant protection of the
mice against the lethal LPS challenge was also observed when LF-33 (100 µg) was given intravenously after intraperitoneal injection of LPS.
Protection was correlated with a reduction in TNF- levels in the
mouse serum. These results demonstrate the endotoxin-neutralizing
capability of LF-33 in vitro and in vivo and its potential use for the
treatment of endotoxin-induced septic shock.
| |
INTRODUCTION |
Endotoxin (lipopolysaccharide
[LPS]) is a constitutive component of the outer membrane of
gram-negative bacteria and is released when the bacteria die or
multiply (29). It is estimated that every year in the United
States, approximately 400,000 patients present with bacterial sepsis,
of which 100,000 ultimately die of septic shock, and about half of
these cases are caused by gram-negative bacteria (26).
Gram-negative sepsis and septic shock primarily result from
endotoxin-induced excessive production and release of inflammatory
cytokines by cells of the immune system, particularly macrophages
(3, 31). Tumor necrosis factor alpha (TNF-) is the
primary mediator of the systemic toxicity of endotoxin (3,
14). Consequently, neutralization of endotoxin represents an
important aspect of a logical, multifaceted approach to treating this
complex clinical syndrome (36). This approach is potentially specific since it does not interfere with the host defense.
Lipid A is the toxic portion of endotoxin (29). Monoclonal
anti-lipid A antibodies have been tested for treating gram-negative sepsis and septic shock, but their clinical efficacy has not been demonstrated consistently (40), probably due to their poor
ability to bind and neutralize endotoxin (41). Newer
developments include identification of synthetic antiendotoxin peptides
mimicking polymyxin B (33) and a number of cationic
antiendotoxin peptides derived from host defense proteins. These
include a recombinant 23-kDa fragment derived from
bactericidal/permeability-increasing protein (10, 21), a
28-mer peptide derived from bee melittin (13), a 33-mer
peptide derived from an 18-kDa cationic antibacterial protein
(18), and synthetic peptides based on the crystal structure of Limulus anti-LPS factor (28).
Lactoferrin is an iron-binding glycoprotein that is synthesized by
mucosal epithelium and neutrophils and released by these cells in
response to inflammatory stimuli (16, 19). It has antimicrobial activities in vitro (19), and lactoferrin
treatment in vivo has been reported to lower the incidence of
gram-negative bacteremia (37). Structurally, it contains a
strongly basic region close to the N terminus which binds to a variety
of anionic biological molecules, including lipid A (1) and
glycosaminoglycans that occur on the surface of most cells and in most
extracellular matrices (20). Lactoferricin H (residues 1 to
47) and lactoferricin B (residues 17 to 41) are released by
pepsinolysis of human and bovine lactoferrin, respectively, and
may have more potent antibacterial activity than the native proteins
(2). A region composed of residues 28 to 34 is reported to
contribute to the high-affinity binding of lactoferrin and
lactoferricin H to endotoxin (6, 39). Lactoferrin and
lactoferricin B have been shown to inhibit the endotoxin-induced
interleukin-6 response in human monocytic cells (23).
Previous studies have established that the N-terminal 33 residues of
human lactoferrin represent the minimal sequence that mediates binding
of the protein to glycosaminoglycans (20). This sequence
contains a cationic head (residues 1 to 6) and tail (residues 28 to 33)
which combine to form the glycosaminoglycan-binding site. In this
study, we sought to assess the endotoxin-neutralizing capacity of a
synthetic peptide, designated LF-33, corresponding to the first 33 residues of the secreted form of human lactoferrin. We measured the
peptide-mediated inhibition of endotoxin-induced Limulus
amebocyte lysate (LAL) coagulation with a sensitive LAL assay
(43) and suppression of endotoxin-induced TNF- secretion by the macrophage cell line RAW 264.7 (17). We also examined the ability of LF-33 to suppress endotoxin-induced TNF- secretion in
the presence of human serum. Finally, we evaluated the protection provided by LF-33 to galactosamine-sensitized mice against a lethal endotoxin challenge.
| |
MATERIALS AND METHODS |
Peptides.
Lactoferrin-derived peptides were synthesized by
conventional Fmoc [N-(9-fluoreny)methoxycarbonyl]
chemistry as described elsewhere (20). The
33-mer peptide (GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP) corresponding to the first 33 residues at the N terminus of human lactoferrin is designated LF-33 (molecular weight [MW], 4,004). The
27-mer peptide, LF-27 (MW, 3,276), corresponds to LF-33 lacking its
N-terminal six residues. Polymyxin B (MW, 1,066; Sigma, St. Louis,
Mo.), an antiendotoxin peptide (4), is used as a reference for comparison throughout this study.
LPS.
Control standard endotoxins from Escherichia
coli O113:H10 and Salmonella abortus equi (Associates
of Cape Cod, Inc., Woods Hole, Mass.) had a potency of 10 endotoxin
units (EU) per ng. LPS (purity >99%) from Neisseria
meningitidis was prepared from the group B strain 6275 in our
laboratory, and its potency was 25 EU/ng. Lipid A from E. coli K-12 (List Biological Laboratories, Inc., Campbell, Calif.)
had a potency of 8.6 EU/ng. The potency of the LPS from
Pseudomonas aeruginosa (Sigma) was 0.12 EU/ng. The potency
of the endotoxin described above was determined with the
Limulus enzyme-linked immunosorbent assay (ELISA)
(43) in comparison with the U.S. Pharmacopeia reference
standard endotoxin EC-5.
Limulus ELISA for determining the ENC50
of antiendotoxin agents.
The Limulus ELISA
is an endotoxin assay based on activation of LAL coagulation by
endotoxin and detection of the generated peptide C immunoreactivity
with an ELISA using a monoclonal antibody to the peptide
(43). Endotoxin was defined to be neutralized when it lost
its ability to activate the LAL enzymes. The 50% endotoxin-neutralizing concentration (ENC50) reflects the
potency of an antiendotoxin agent; a low ENC50 indicates
high potency.
Briefly, 25 µl of endotoxin solution (200 EU/ml) was mixed with an
equal volume of test materials in a series of twofold dilutions in 0.15 M NaCl in a sterile 96-well tissue culture plate (Nunc A/S, Roskilde,
Denmark) and incubated at 37°C for 1 h in a dry-air incubator.
The reaction mixtures were diluted 1,000-fold with endotoxin-free
water. The endotoxin activity was then quantified with the use of the
Limulus ELISA (43). In the Limulus
ELISA, endotoxin activated the LAL coagulation at concentrations below 1 pg or 0.01 EU per ml (43). The high sensitivity of the
assay allowed for very low levels of the endotoxin activity to be
detected. Following incubation of endotoxin with test materials, a
1,000-fold dilution was introduced to eliminate any potential effects
of the test materials on the LAL enzyme system. Neither serum nor any
of these materials interfered with the enzymatic cascade of the LAL
assay itself after a 1,000-fold dilution from their highest concentrations used in this study. For each assay, the LAL-endotoxin reaction was carried out under optimal conditions with a linear relationship between the concentration of endotoxin and the optical density at 490 nm. A sigmoid curve was usually obtained between optical
density at 490 nm and the logarithmic concentration of an antiendotoxin
agent. The concentration corresponding to the midpoint of the curve was
designated ENC50.
Endotoxin-induced TNF- secretion by RAW 264.7 cells.
The
murine macrophage cell line RAW 264.7 was obtained from the American
Type Culture Collection (ATCC, Rockville, Md.) and maintained as
described previously (17). The concentration of endotoxin in
all buffers and media was controlled to below 0.1 EU/ml. The following
protocol was essentially described before and used here with minor
modifications (17). Each well of a 96-well tissue culture
plate was seeded with 150 µl of RAW 264.7 cells at 106
cells per ml of Dulbecco minimal essential medium (Life
Technologies, Gaithersburg, Md.) supplemented with 10% heat-treated
fetal bovine serum (Life Technologies), 25 mM HEPES (pH 7.3),
penicillin (60 U/ml), and streptomycin (60 µg/ml). After
overnight incubation at 37°C in a 6% CO2
incubator, the medium was aspirated and the cells were washed with
three changes of endotoxin-free Hank's balanced salt solution (HBSS;
Life Technologies) supplemented with 25 mM HEPES (pH 7.3). Control
endotoxin (10 ng/ml) and test materials were prepared in HBSS-HEPES.
After incubation in a 37°C water bath for 1 h, 0.2 ml of these
solutions was added in triplicate to each well and incubated at 37°C
for 6 h. The supernatants were then collected and stored at
70°C before the measurement of TNF- activity. Controls included
HBSS-HEPES and test materials in the absence of endotoxin. TNF-
activity of the medium and test materials was below 160 pg/ml. The
human serum used in some experiments was a pool from normal donors.
The cytotoxic assay.
TNF- activity in the culture
supernatant was determined on the basis of its cytotoxicity for the
mouse fibrosarcoma cell line WEHI 164 (ATCC). We observed that this
cell line was fourfold more sensitive to TNF- than the commonly used
L929 fibroblast cells, and the sensitivity was further increased
fivefold by inclusion of actinomycin D (Life Technologies) in the
medium (32). In this assay, the concentration of active
TNF- was correlated with cell death resulting from exposure to
TNF-. Cell death was measured colorimetrically with the viable dye
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
method (24). The specificity of the assay was verified by
using a rabbit anti-mouse TNF- antibody (Genzyme, Inc., Cambridge, Mass.). The antibody at a 1:100 dilution completely eliminated the
cytotoxicity in the culture supernatant of the RAW 264.7 cells stimulated by endotoxin and in the mouse sera collected 1 h after intraperitoneal (i.p.) injection of endotoxin.
Briefly, 96-well tissue culture plates were seeded with 100 µl of
WEHI 164 cells (5 × 10
4 cells) in RPMI 1640 medium
(Life Technologies) containing 10%
heat-treated fetal bovine serum, 25 mM HEPES (pH 7.3), penicillin
(60 U/ml), streptomycin (60 µg/ml), and
actinomycin D (4 µg/ml).
After a 2-h incubation at 37°C in a 6%
CO
2 incubator, 10 µl of
twofold serially diluted samples
(culture supernatants) or standards
(murine recombinant TNF-
;
Genzyme) was added to each well and
the wells were incubated for
20 h. Cell viability was then determined
by the addition of 10 µl of MTT (thiazolyl blue; Sigma) stock
solution (5 mg/ml in saline)
to each well, and the incubation
was allowed to continue for 6 h.
One hundred eighty microliters
of acid-isopropanol (containing 40 mM
HCl) was added to dissolve
the generated dark blue crystals. The plate
was read at 570 nm
with a reference of 630 nm in a microplate reader.
The amount
of TNF-
that led to 50% killing of the seeded cells was
defined
as 1 U, equivalent to approximately 15 pg of recombinant
TNF-
under the present condition. A standard curve was obtained
by
incubating known amounts of the recombinant TNF-
with the
WEHI
164
cells.
To exclude any potential cytotoxicity of LF-33, the procedure described
above was followed except that WEHI 164 cells were
replaced by RAW
264.7 cells and the concentration of cells seeded
in each well was
1.5 × 10
5 per 150 µl of medium to mimic the
conditions in the stimulation
experiment. At the highest concentration
of LF-33 (10 µM) used
in this study, no cytotoxicity to RAW 264.7 cells was
detected.
Galactosamine-sensitized mouse model.
Mice are typically
resistant to endotoxin. However, the sensitivity of mice to endotoxin
can be enhanced more than 1,000-fold by coinjection with a
liver-specific inhibitor, galactosamine (11, 12). An
essential feature of this in vivo model is that systemically released
TNF- causes liver damage due to TNF--mediated liver cell death,
which can be scored by measuring lethality. In our study, i.p.
injection of 125 ng of E. coli LPS together with 15 mg of
galactosamine hydrochloride (Sigma) in 0.5 ml of 0.15 M NaCl induced
nearly 100% lethality in 8- to 10-week-old female NIH/Swiss mice (body
weight, 20 to 25 g/mouse). LF-33 was either injected intravenously
(i.v.) through tail veins 10 min after the i.p. injection of the
LPS-galactosamine mixture or coinjected i.p. with LPS and
galactosamine. Lethality was observed for 72 h after injection. In
experiments involving measurement of the TNF- level in serum, blood
samples were collected in serum separator tubes (Becton Dickinson,
Rutherford, N.J.) 60 to 90 min postinjection, and sera were obtained
after centrifugation. The TNF- level in serum was measured by the
cytotoxic assay described above. The peak TNF- level in serum was
found between 60 and 90 min after i.p. injection of LPS.
Statistics.
We performed all endotoxin and TNF-
measurements in triplicate in each experiment. At least two independent
experiments were performed for all data. Values are given as the
mean ± standard deviation (SD) and were compared by using the
unpaired Student t test. Lethality is compared by use of
Fisher's exact test.
| |
RESULTS |
Inhibition of endotoxin-induced LAL coagulation.
ENC50 values of each antiendotoxin agent against
lipid A and four different types of LPS are listed in Table
1. A low ENC50 indicates high
potency of endotoxin neutralization. The potency of each antiendotoxin
agent varied depending on the type of endotoxin. LF-33 was more potent
than polymyxin B, on a molar basis, at neutralizing all forms of
endotoxin tested. In contrast, LF-27 was approximately 10-fold-less
potent than LF-33 at neutralizing lipid A and E. coli LPS
and had no detectable activity against the other three LPSs. Human
serum showed various degrees of inhibition of endotoxin-induced LAL
coagulation but had no effect on lipid A (Table 1).
Suppression of endotoxin-induced TNF- secretion by LF-33.
RAW 264.7 cells secrete TNF- upon exposure to endotoxin
(32). A linear relationship between TNF- secretion and
endotoxin concentration was observed at endotoxin concentrations below
20 ng/ml for lipid A and the various LPSs used in this study, and a
concentration of 10 ng/ml of endotoxin was selected for the TNF--inducing experiments. Mixing endotoxin with increasing
concentrations of LF-33 resulted in a dose-dependent suppression of
endotoxin-induced TNF- secretion (Fig.
1). Similar to the results of the LAL
assay, the potency of LF-33 varied depending on the type of endotoxin. The LF-33 concentrations needed to suppress TNF- secretion induced by endotoxin (10 ng/ml) by 50% were approximately 0.01 µM for E. coli LPS and lipid A, 0.1 µM for LPS from P. aeruginosa, and 0.5 µM for LPS from S. abortus equi
and N. meningitidis. The effects of LF-27, polymyxin B,
and human serum on endotoxin-induced TNF- secretion are shown in
Table 2 for a comparison. LF-33 exhibited a slightly higher potency than polymyxin B in suppressing TNF- secretion induced by different types of endotoxin, whereas an equimolar
concentration of LF-27 or 10% human serum had no effect on
endotoxin-induced TNF- secretion.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Dose-dependent suppression by LF-33 of endotoxin-induced
TNF- secretion by RAW 264.7 cells. Endotoxin at 10 ng/ml was
incubated at 37°C for 1 h with LF-33 at the concentrations
indicated before being exposed to RAW 264.7 cells. All data were the
means of triplicates in representative experiments. S. abortus,
S. abortus equi; P. aer, P. aeruginosa;
N. men, N. meningitidis.
|
|
Effect of human serum on the LF-33 suppression of endotoxin-induced
TNF- secretion.
To test the suppression of endotoxin-induced
TNF- secretion under more physiological conditions, LF-33 or
polymyxin B was added to human serum (final concentration, 10%) before
the addition of endotoxin. As shown in Table
3, the suppressive effect of the peptides
was attenuated substantially in the presence of 10% human serum,
although the serum effect could be overcome by increasing the
concentration of LF-33 (Table 3 and Fig.
2). However, if the peptide was mixed
with endotoxin 5 min before the addition of serum, the effect of the
serum on the neutralization of endotoxin by the peptides was greatly
reduced (Table 4).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Suppression by antiendotoxin peptides of
endotoxin-induced TNF- secretion by RAW 264.7 cells in culture
medium containing 10% human seruma
|
|
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 2.
Dose-dependent suppression by LF-33 of endotoxin-induced
TNF- secretion by RAW 264.7 cells in human serum. E. coli
LPS at 10 ng/ml was incubated at 37°C for 1 h with human serum
and LF-33 at the concentrations indicated before being exposed to RAW
264.7 cells. All data were the means of triplicates in representative
experiments.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Suppression by antiendotoxin peptides of
endotoxin-induced TNF- secretion by RAW 264.7 cells in culture
medium containing 10% human serum: effect of the mixing sequence
with serum
|
|
Effect of LF-33 on endotoxin-induced lethality and the TNF-
level in serum in the galactosamine-sensitized mouse model.
Injection of 125 ng of E. coli LPS per animal by the i.p.
route induced nearly 100% lethality in the galactosamine-sensitized mice. As shown in Table 5, the
endotoxin-induced lethality was dramatically reduced by injecting
LF-33. Small amounts of LF-33 (2.5 µg per animal), when injected
simultaneously with endotoxin, reduced the lethality from 93% (14 of
15 animals dead) to 6% (1 of 15 animals dead). In addition, LF-33 also
significantly reduced the lethality when injected i.v. 10 min
subsequent to the i.p. injection of endotoxin (Table 5), although a
40-fold-greater amount of LF-33 was required. The protection was
correlated with the reduction of the TNF- level in mouse serum
(Table 5).
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Protection of animals against the lethality of LPS
provided by LF-33 in the galactosamine-sensitized mouse model
|
|
| |
DISCUSSION |
Current treatments for gram-negative sepsis and septic shock
rely on antibiotics to control the infection and intensive-care support to correct the dysfunction of cardiovascular, respiratory, and
other organ systems. Research into the pathogenesis of this fatal
clinical syndrome points to endotoxin as a principal pathogenic factor
and TNF- as a primary mediator of endotoxicity. Although a
number of antiendotoxin proteins and peptides have been reported (10, 13, 18, 28, 33), there is still no antiendotoxin agent
licensed for clinical use to supplement the current therapy. Our study
establishes that the human lactoferrin-derived peptide LF-33 possesses
a potent endotoxin-neutralizing capacity in vitro and in vivo as shown
by its ability to suppress endotoxin-induced LAL coagulation and
TNF- secretion by RAW 264.7 cells and to protect animals from a
lethal endotoxin challenge.
Because the lipid A portion of endotoxin is responsible for the
activation of LAL (15), stimulation of TNF- secretion
(29), and lethality in mice (29), LF-33 likely
exerts its antiendotoxin actions by binding to the lipid A portion and
consequently blocking the biological effects of endotoxin. This is
supported by its direct neutralization of lipid A in addition to four
different types of endotoxin (Table 1). The only difference between the sequence of LF-33 and LF-27 is that LF-27 lacks the first six residues
(GRRRRS) at the N terminus of LF-33. Remarkably, this deletion led to a
dramatic loss of the endotoxin-neutralizing capacity of the peptide in
the LAL assay and complete loss in the TNF- bioassay (Tables 1 and
2), indicating the importance of the cationic head of LF-33 in
neutralizing endotoxin. This cluster of basic residues has previously
been shown to be required for the binding of lactoferrin to other
anionic molecules, including glycosaminoglycans (20).
Because most known antiendotoxin peptides are cationic in nature and
both the lipid A and oligosaccharide core portion of LPS are anionic,
electrostatic forces may contribute to the binding of endotoxin and the
neutralizing cationic peptides. Indeed, the presence of additional
ethanolamine groups (positively charged) in the lipid A portion of LPS
from N. meningitidis and S. abortus
equi, but their absence in LPS from E. coli and
P. aeruginosa (27, 30), is consistent with
the observed greater potency of LF-33 and polymyxin B in
suppressing TNF- secretion induced by endotoxin from the
latter types of bacteria compared to the former types (Table 2 and Fig.
1).
LF-27 showed some detectable antiendotoxin activity against
lipid A and E. coli LPS but was essentially inactive
against the other three types of LPS examined in the LAL assay (Table
1) and completely inactive in inhibiting the TNF- production induced by all five of the endotoxins tested (Table 2). These apparent discrepancies likely reflect the difference in affinities of LF-27 for
the different types of endotoxins and the difference in sensitivities and complexities of the two assay systems. The LAL assay is more than
100 times more sensitive than the TNF- bioassay for detecting endotoxin and therefore would allow the low-affinity binding between LF-27 and endotoxin to be detected.
The endotoxin-neutralizing activity of human serum has been
observed previously in the Limulus assay (8,
42) and confirmed by our study, as shown in Table 1 and Fig. 2.
Noticeably, much lower concentrations (<2.5% compared to >10%) of
human serum are needed to neutralize the endotoxin activity measured in
the Limulus assay (Table 1) than in the cell assay
(endotoxin-induced TNF- secretion by RAW 264.7 cells)
(Fig. 2 and Table 2). Binding of serum proteins to endotoxin
neutralizes the endotoxin activity in the Limulus assay by
preventing endotoxin from activating LAL (8). However, this
is not necessarily true in the cell assay. It is now known that the
initial step in the endotoxin-induced cellular response is the binding
of endotoxin to serum LPS-binding protein (LBP). Endotoxin in this
complexed form is much more effective than free endotoxin in binding to
CD14, a glycosylphosphatidylinositol-anchored membrane
protein on myeloid cells (22, 34), and subsequently triggering and enhancing the production and release of inflammatory mediators including TNF- (38). Thus, in the cell assay,
LBP in the serum can counteract the endotoxin-neutralizing activity of
other serum LPS-binding molecules such as lipoproteins. In addition, since EDTA potentiates and divalent cations
(Mg2+ and Ca2+) reduce the
endotoxin-neutralizing capacity of human serum (25, 42), the presence of divalent cations in the incubation
medium in the cell assay should further decrease the
endotoxin-neutralizing capacity of the serum.
LF-33 appears to be more potent than polymyxin B at suppressing
endotoxin-induced LAL coagulation and TNF- secretion by RAW 264.7 cells under serum-free conditions (Table 1 and 2). This suggests
that LF-33 may have a greater intrinsic capacity to neutralize endotoxin than polymyxin B does. In the presence of human serum, however, their antiendotoxin potencies become more similar since, although human serum significantly attenuates the
endotoxin-neutralizing capacity of both peptides, it has a greater
blocking effect on LF-33 (compare Tables 2 and 3). A similar effect
of human serum has also been observed with other cationic antiendotoxin
peptides such as Limulus anti-LPS factor-derived peptides
(28) and a synthetic antiendotoxin peptide (5) in
different assay systems. This appears to be due to the interaction of
these peptides with serum proteins that effectively reduce the
availability of the peptides for binding to endotoxin. Consistent with
this explanation is our observation that mixing LF-33 with serum before
endotoxin dramatically reduces the ability of the peptide to suppress
endotoxin-induced TNF- secretion (Table 4). For example, serum LBP
may compete with LF-33 in binding to endotoxin, since lactoferrin has
recently been shown to inhibit the endotoxin interaction with CD14 by
competing for the binding of endotoxin to LBP (7). The
blocking effect of human serum may partly explain the inability of a
low dose of LF-33 to protect mice against the lethality of endotoxin
when the peptide was injected into blood after i.p. injection of
endotoxin, whereas almost complete protection was observed when they
were mixed before i.p. injection (Table 5). Thus, increasing the dose of LF-33 could overcome the serum blocking effect (Table 3 and Fig. 2)
and protect the mice against the lethality of endotoxin even when the
peptide was injected separately from endotoxin (Table 5).
In conclusion, we have shown that a novel synthetic peptide
representing the minimal sequence that mediates binding of human lactoferrin to glycosaminoglycans, LF-33, has potent
endotoxin-neutralizing properties in vitro and in vivo against lipid A
and different types of LPS. The endotoxin-neutralizing capacity of
LF-33 is greater than that of polymyxin B when tested in serum-free
media and comparable to that of polymyxin B in the presence of human serum. In a separate study, LF-33 has also been found to be
bactericidal to various gram-negative bacteria (20a). The
dual properties of LF-33 in neutralizing endotoxin and killing bacteria
may present potential advantages over the conventional antibiotics such
as 
-lactams and quinolones, since these antibiotics are known to promote endotoxin release but have no endotoxin-neutralizing activity and can thus cause endotoxemia during antimicrobial therapy (9, 35). Considering that cationic peptides generally have a short half-life in blood while clinical endotoxemia can be intermittent and
recurrent, we are currently investigating agents that can overcome the
serum attenuation of the anti-LPS potency of these peptides and are
designing analogues with enhanced half-life and efficacy in blood.
| |
ACKNOWLEDGMENTS |
We thank Claus Koch of the Statens Seruminstitut, Copenhagen,
Denmark, for providing us with the essential regents for the Limulus ELISA and Carl E. Frasch, Che-Hung Lee, and Karin
Elkins from the U.S. Food and Drug Administration for their valuable suggestions.
Gui-Hang Zhang was supported by a Visiting Fellowship from the Fogarty
International Center, National Institutes of Health, and David M. Mann
was supported by a grant (award AI39691) from the National Institute of
Allergy and Infectious Diseases, National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Plasma
Derivatives Department, J. H. Holland Laboratory, American
Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Phone: (301)
738-0545. Fax: (301) 738-0794. E-mail:
zhanggu{at}usa.redcross.org.
Editor:
R. N. Moore
| |
REFERENCES |
| 1.
|
Appelmelk, B. J.,
Y.-Q. An,
M. Geerts,
B. G. Thijs,
H. A. DeBoer,
D. M. MacLaren,
J. DeGraaff, and J. H. Nuijens.
1994.
Lactoferrin is a lipid A-binding protein.
Infect. Immun.
62:2628-2632[Abstract/Free Full Text].
|
| 2.
|
Bellamy, W.,
M. Takase,
K. Yamauchi,
H. Wakabayashi,
K. Kawase, and M. Tomita.
1992.
Identification of the bactericidal domain of lactoferrin.
Biochim. Biophys. Acta
1121:130-136[Medline].
|
| 3.
|
Beutler, B., and A. Cerami.
1988.
Tumor necrosis, cachexia, shock, and inflammation: a common mediator.
Annu. Rev. Biochem.
57:505-518[Medline].
|
| 4.
|
Cooperstock, M. S.
1974.
Inactivation of endotoxin by polymyxin B.
Antimicrob. Agents Chemother.
6:422-425[Abstract/Free Full Text].
|
| 5.
|
Demistri, M. T.,
M. Velucchi,
L. Bracci,
A. Rustici,
M. Porro,
P. Villa, and P. Ghezzi.
1996.
Inhibition of LPS-induced systemic and local TNF production by a synthetic anti-endotoxin peptide (SAEP-2).
J. Endotoxin Res.
3:445-454.
|
| 6.
|
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 O55:B5 lipopolysaccharide.
Biochem. J.
312:839-845.
|
| 7.
|
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].
|
| 8.
|
Emancipator, K.,
G. Csako, and R. J. Elin.
1992.
In vivo inactivation of bacterial endotoxin by human lipoproteins and apolipoproteins.
Infect. Immun.
60:596-601[Abstract/Free Full Text].
|
| 9.
|
Evans, M. E., and M. Pollack.
1993.
Effect of antibiotic class and concentration on the release of lipopolysaccharide from Escherichia coli.
J. Infect. Dis.
167:1336-1343[Medline].
|
| 10.
|
Fisher, C. J.,
M. N. Marra,
J. E. Palardy,
C. R. Marchbanks,
R. W. Scott, and S. M. Opal.
1994.
Human neutrophil bactericidal/permeability-increasing protein reduces mortality rate from endotoxin challenge: a placebo-controlled study.
Crit. Care Med.
22:553-558[Medline].
|
| 11.
|
Freudenberg, M. A., and C. Galanos.
1991.
Tumor necrosis factor alpha mediates lethal activity of killed gram-negative and gram-positive bacteria in D-galactosamine-treated mice.
Infect. Immun.
59:2110-2115[Abstract/Free Full Text].
|
| 12.
|
Galanos, C.,
M. A. Freudenberg, and W. Reutter.
1979.
Galactosamine-induced sensitization to the lethal effects of endotoxin.
Proc. Natl. Acad. Sci. USA
76:5939-5943[Abstract/Free Full Text].
|
| 13.
|
Gough, M.,
R. E. W. Hancock, and N. M. Kelly.
1996.
Antiendotoxin activity of cationic peptide antimicrobial agents.
Infect. Immun.
64:4922-4927[Abstract].
|
| 14.
|
Heumann, D.,
D. Le Roy, and M. P. Glauser.
1996.
Contribution of TNF and of LPS in endotoxemic shock in mice: a reappraisal.
J. Endotoxin Res.
3:87-92.
[Abstract/Free Full Text] |
| 15.
|
Iwanaga, S.,
T. Miyata,
F. Tokunaga, and T. Muta.
1992.
Molecular mechanism of hemolymph clotting system in Limulus.
Thromb. Res.
68:1-32[Medline].
|
| 16.
|
Jeremy, B.
1995.
Lactoferrin: a multifunctional immunoregulatory protein?
Immunol. Today
16:417-419[Medline].
|
| 17.
|
Kelly, N. M.,
L. Young, and A. Cross.
1991.
Differential induction of tumor necrosis factor by bacteria expressing rough and smooth lipopolysaccharide phenotypes.
Infect. Immun.
59:4491-4496[Abstract/Free Full Text].
|
| 18.
|
Larrick, J. W.,
M. Hirata,
R. F. Balint,
J. Lee,
J. Zhong, and S. C. Wright.
1995.
Human CAP18: a novel antimicrobiol lipopolysaccharide-binding protein.
Infect. Immun.
63:1291-1297[Abstract].
|
| 19.
|
Lonnerdal, B., and S. Iyer.
1995.
Lactoferrin: molecular structure and biological function.
Annu. Rev. Nutr.
15:93-110[Medline].
|
| 20.
|
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].
|
| 20a.
| Mann, D. M., et al. Submitted for publication.
|
| 21.
|
Marra, M. N.,
M. B. Thornton,
J. L. Snable,
C. G. Wilde, and R. W. Scott.
1994.
Endotoxin-binding and -neutralizing properties of recombinant bactericidal/permeability-increasing protein and monoclonal antibodies HA-1A and E5.
Crit. Care Med.
22:559-565[Medline].
|
| 22.
|
Mathison, J. C.,
P. S. Tobias,
E. Wolfson, and R. J. Ulevitch.
1992.
Plasma LPS-binding protein, a key component in macrophage recognition of gram-negative LPS.
J. Immunol.
194:200-206.
|
| 23.
|
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].
|
| 24.
|
Mosmann, T.
1983.
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay.
J. Immunol. Methods
65:55-63[Medline].
|
| 25.
|
Ogata, M.,
M. F. Fletcher,
M. Kloczewiak,
P. M. Loiselle,
E. M. Zanzot,
M. W. Vermeulen, and H. S. Warren.
1997.
Effect of anticoagulants on binding and neutralization of lipopolysaccharide by the peptide immunoglobulin conjugate CAP18106-138-immunoglobulin G in whole blood.
Infect. Immun.
65:2160-2167[Abstract].
|
| 26.
|
Parrillo, J. E.
1996.
Shock syndromes related to sepsis, p. 496-501.
In
J. C. Bennett, and F. Plum (ed.), Cecil textbook of medicine, 20th ed. The W. B. Saunders Co., Philadelphia, Pa.
|
| 27.
|
Poolman, J. T.
1995.
Development of a meningococcal vaccine.
Infect. Agents Dis.
4:13-28[Medline].
|
| 28.
|
Reid, C.,
C. Wahl,
T. Miethke,
G. Wellnhofer,
C. Landgraft,
J. Schneider-Mergener, and A. Hoess.
1996.
High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor.
J. Biol. Chem.
271:28120-28127[Abstract/Free Full Text].
|
| 29.
|
Rietschel, E. T.,
T. Kirikae,
F. U. Schade,
A. J. Ulmer,
O. Holst,
H. Brade,
G. Schmidt,
U. Mamat,
H.-D. Grimmecke,
S. Kusumoto, and U. Zaahringer.
1993.
The chemical structure of bacterial endotoxin in relation to bioactivity.
Immunobiology
187:169-190[Medline].
|
| 30.
|
Rietschel, E. T.,
H.-W. Wollenweber,
H. Brade,
U. Zaahringer,
B. Lindner,
U. Seydel,
H. Bradaczek,
G. Barnickel,
H. Labischinski, and P. Giesbrecht.
1984.
Structure and conformation of the lipid A component of lipopolysaccharides, p. 187-220.
In
E. T. Rietschel (ed.), Handbook of endotoxin, vol. 1. Chemistry of endotoxin. Elsevier Science Publishers B. V., New York, N.Y.
|
| 31.
|
Rosenstreich, D. L., and S. N. Vogel.
1980.
Central role of macrophages in the host response to endotoxin, p. 11-15.
In
D. Schlessinger (ed.), Microbiology-1980. American Society for Microbiology, Washington, D.C.
|
| 32.
|
Ruff, M. R., and G. E. Gifford.
1981.
Tumor necrosis factor.
Lymphokines
2:235-272.
|
| 33.
|
Rustici, A.,
M. Velucchi,
R. Faggioni,
M. Sironi,
P. Ghezzi,
S. Quataert,
B. Green, and M. Porro.
1993.
Molecular mapping and detoxification of the lipid A binding site by synthetic peptides.
Science
259:361-365[Abstract/Free Full Text].
|
| 34.
|
Schumann, R. R.
1992.
Function of LPS-binding protein (LBP) and CD14, the receptor for LPS/LBP complex: a short review.
Res. Immunol.
143:11-15[Medline].
|
| 35.
|
Shenep, J. L.,
R. P. Barton, and K. A. Mogan.
1985.
Role of antibiotic class in the rate of liberation of endotoxin during therapy for experimental gram-negative bacterial sepsis.
J. Infect. Dis.
151:1012-1018[Medline].
|
| 36.
|
Tanamoto, K.-I.
1997.
Novel strategies to attenuate the effect of lipopolysaccharide on the immune response.
Curr. Opin. Infect. Dis.
10:177-182.
|
| 37.
|
Trumpler, U.,
P. W. Straub, and A. Rosemund.
1989.
Antibacterial prophylaxis with lactoferrin in neutropenic patients.
Eur. J. Clin. Microbiol. Infect. Dis.
8:310-313[Medline].
|
| 38.
|
Ulevitch, R. J., and P. S. Tobias.
1995.
Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.
Annu. Rev. Immunol.
13:437-457[Medline].
|
| 39.
|
van Berkel, P. H. C.,
M. E. J. Geerts,
H. A. van Veen,
M. Mericskay,
H. A. de Boer, and J. H. Nuijens.
1997.
N-terminal stretch Arg2, Arg3, Arg4, Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.
Biochem. J.
328:145-151.
|
| 40.
|
Verhoef, J.,
W. M. N. Hustinx,
H. Frasa, and A. I. M. Hoepelman.
1996.
Issues in the adjunct therapy of severe sepsis.
J. Antimicrob. Chemother.
38:167-182[Abstract/Free Full Text].
|
| 41.
|
Warren, H. S.,
S. F. Amato,
C. Fitting,
K. M. Black,
P. M. Loiselle,
M. S. Pasternack, and J.-M. Cavaillon.
1993.
Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.
J. Exp. Med.
177:89-97[Abstract/Free Full Text].
|
| 42.
|
Zhang, G. H.,
L. Baek,
T. Bertelsen, and C. Koch.
1995.
Quantification of the endotoxin-neutralizing capacity of serum and plasma.
APMIS
103:721-730[Medline].
|
| 43.
|
Zhang, G. H.,
L. Baek,
P. E. Nielsen,
O. Buchardt, and C. Koch.
1994.
Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with a monoclonal antibody against Limulus peptide C.
J. Clin. Microbiol.
32:416-422[Abstract/Free Full Text].
|
Infection and Immunity, March 1999, p. 1353-1358, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hunter, H. N., Demcoe, A. R., Jenssen, H., Gutteberg, T. J., Vogel, H. J.
(2005). Human Lactoferricin Is Partially Folded in Aqueous Solution and Is Better Stabilized in a Membrane Mimetic Solvent. Antimicrob. Agents Chemother.
49: 3387-3395
[Abstract]
[Full Text]
-
McMichael, J. W., Roghanian, A., Jiang, L., Ramage, R., Sallenave, J.-M.
(2005). The Antimicrobial Antiproteinase Elafin Binds to Lipopolysaccharide and Modulates Macrophage Responses. Am. J. Respir. Cell Mol. Bio.
32: 443-452
[Abstract]
[Full Text]
-
Santamaria, C., Larios, S., Quiros, S., Pizarro-Cerda, J., Gorvel, J.-P., Lomonte, B., Moreno, E.
(2005). Bactericidal and Antiendotoxic Properties of Short Cationic Peptides Derived from a Snake Venom Lys49 Phospholipase A2. Antimicrob. Agents Chemother.
49: 1340-1345
[Abstract]
[Full Text]
-
Silverstein, R.
(2004). Review: D-Galactosamine lethality model: scope and limitations. Innate Immunity
10: 147-162
[Abstract]
-
Andra, J., Koch, M. H. J., Bartels, R., Brandenburg, K.
(2004). Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin. Antimicrob. Agents Chemother.
48: 1593-1599
[Abstract]
[Full Text]
-
Ochoa, T. J., Noguera-Obenza, M., Ebel, F., Guzman, C. A., Gomez, H. F., Cleary, T. G.
(2003). Lactoferrin Impairs Type III Secretory System Function in Enteropathogenic Escherichia coli. Infect. Immun.
71: 5149-5155
[Abstract]
[Full Text]
-
Van Amersfoort, E. S., Van Berkel, T. J. C., Kuiper, J.
(2003). Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock. Clin. Microbiol. Rev.
16: 379-414
[Abstract]
[Full Text]
-
Caccavo, D., Pellegrino, N. M., Altamura, M., Rigon, A., Amati, L., Amoroso, A., Jirillo, E.
(2002). Review: Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application. Innate Immunity
8: 403-417
[Abstract]
-
Teng, C. T., Beard, C., Gladwell, W.
(2002). Differential Expression and Estrogen Response of Lactoferrin Gene in the Female Reproductive Tract of Mouse, Rat, and Hamster. Biol. Reprod.
67: 1439-1449
[Abstract]
[Full Text]
-
Scott, M. G., Davidson, D. J., Gold, M. R., Bowdish, D., Hancock, R. E. W.
(2002). The Human Antimicrobial Peptide LL-37 Is a Multifunctional Modulator of Innate Immune Responses. J. Immunol.
169: 3883-3891
[Abstract]
[Full Text]
-
Kim, H. S., Cho, J. H., Park, H. W., Yoon, H., Kim, M. S., Kim, S. C.
(2002). Endotoxin-Neutralizing Antimicrobial Proteins of the Human Placenta. J. Immunol.
168: 2356-2364
[Abstract]
[Full Text]
-
Nibbering, P. H., Ravensbergen, E., Welling, M. M., van Berkel, L. A., van Berkel, P. H. C., Pauwels, E. K. J., Nuijens, J. H.
(2001). Human Lactoferrin and Peptides Derived from Its N Terminus Are Highly Effective against Infections with Antibiotic-Resistant Bacteria. Infect. Immun.
69: 1469-1476
[Abstract]
[Full Text]
-
Lupetti, A., Paulusma-Annema, A., Welling, M. M., Senesi, S., van Dissel, J. T., Nibbering, P. H.
(2000). Candidacidal Activities of Human Lactoferrin Peptides Derived from the N Terminus. Antimicrob. Agents Chemother.
44: 3257-3263
[Abstract]
[Full Text]
-
Cirioni, O., Giacometti, A., Barchiesi, F., Scalise, G.
(2000). Inhibition of growth of Pneumocystis carinii by lactoferrins alone and in combination with pyrimethamine, clarithromycin and minocycline. J Antimicrob Chemother
46: 577-582
[Abstract]
[Full Text]
-
Scott, M. G., Rosenberger, C. M., Gold, M. R., Finlay, B. B., Hancock, R. E. W.
(2000). An {alpha}-Helical Cationic Antimicrobial Peptide Selectively Modulates Macrophage Responses to Lipopolysaccharide and Directly Alters Macrophage Gene Expression. J. Immunol.
165: 3358-3365
[Abstract]
[Full Text]
-
TAN, N. S., HO, B., DING, J. L.
(2000). High-affinity LPS binding domain(s) in recombinant factor C of a horseshoe crab neutralizes LPS-induced lethality. FASEB J.
14: 859-870
[Abstract]
[Full Text]
-
Iwagaki, A., Porro, M., Pollack, M.
(2000). Influence of Synthetic Antiendotoxin Peptides on Lipopolysaccharide (LPS) Recognition and LPS-Induced Proinflammatory Cytokine Responses by Cells Expressing Membrane-Bound CD14. Infect. Immun.
68: 1655-1663
[Abstract]
[Full Text]
Top
Abstract
Introduction
