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CUTTING EDGE |
*
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; and
Department of General Surgery, Maastricht University, Maastricht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by the RAW 264.7 macrophage cell line. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6,
and other inflammatory mediators (2, 3, 4, 5, 6, 7, 8, 9). Moreover, studies
in mice have shown that cationic peptides can block endotoxin-induced
TNF- release and reduce the mortality associated with endotoxemia in
the galactosamine-sensitized mouse model (2, 4, 6, 9).
Although several cationic peptides have been shown to bind LPS
(2, 7, 10), little is known about how they block the
biological effects of LPS.
The mechanism by which LPS activates macrophages is now understood in
some detail. LPS binding protein
(LBP),3 an acute-phase
reactant that is present in the blood, binds LPS, extracts it from
micelles, and transfers it to CD14, a protein that exists as a soluble
form in blood and as a GPI-linked molecule on the surface of monocytes
and macrophages. LPS·CD14 complexes are thought to initiate
intracellular signaling reactions by binding to Toll-like receptors
(TLRs) on macrophages and other cells (11). TLR4 appears
to be required for LPS to initiate signaling and to induce inflammatory
responses. Inbred strains of mice with loss-of-function mutations in
TLR4 do not respond to LPS (12, 13). LPS·CD14 complexes
activate the NF-
B transcription factor as well as the extracellular
signal-regulated kinase, c-Jun N-terminal kinase, and p38
mitogen-activated protein kinases, all of which mediate the production
of inflammatory cytokines (11, 14, 15, 16). By facilitating
the initial step in LPS signaling, the binding of LPS to CD14, LBP
greatly enhances the efficiency of LPS signaling such that 100-fold
less LPS is required to trigger cytokine secretion
(17).
Because cationic antimicrobial peptides that block LPS-induced
macrophage activation can bind to LPS (2, 4, 5, 10, 18, 19, 20), we hypothesized that these cationic peptides act by
blocking the binding of LPS to LBP. To test this, we used an ELISA-type
assay that measures the ability of biotinylated LPS to bind to
immobilized LBP. Using this assay, we analyzed the ability of
structurally diverse cationic peptides to block the binding of LPS to
LBP. We show that peptides belonging to all of the major structural
groups of antimicrobial peptides can block the binding of LPS to LBP in
the assay. In general, the relative ability of the different peptides
to block the binding of LPS to LBP in vitro correlated with their
ability to block LPS-induced production of TNF- by RAW 264.7 cells.
Thus, the ability of cationic peptides to block macrophage activation
by LPS may be due in large part to their ability to block the binding
of LPS to LBP and prevent the LBP-mediated transfer of LPS to
CD14.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli O55:B5 LPS was purchased from Sigma
(St. Louis, MO) and biotinylated using biotin-LC-hydrazide (Pierce,
Rockford, IL) as described previously (21). CEMA, CP29,
CP208, bactenecin (Bac) 2A-NH2, indolicidin, and
CP11CN were synthesized at the Nucleic Acid/Protein Synthesis Unit at
the University of British Columbia as described previously
(7). Gramicidin S and human neutrophil peptide-1 (HNP-1)
were purchased from Sigma. Human 
-defensin-2 (HBD-2) was kindly
provided by Dr. T. Ganz (University of California, Los Angeles, CA).
The amino acid sequences of these peptides are shown in Table I
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Chineses hamster ovary cells expressing human recombinant LBP were a gift from Dr. P. Tobias. The recombinant LBP was purified from the culture medium as described previously (21) and its concentration determined by ELISA (22).
Measurement of LPS-LBP interactions
The anti-LBP mAb HM14 (21), which recognizes human LBP as well as LBP·LPS complexes, was diluted to 10 µg/ml in PBS and adsorbed onto 96-well Nunc MaxiSorp ELISA plates (Nunc, Rochester, NY) overnight at 4°C. Plates were blocked at room temperature for 1 h with PBS/1% BSA and washed with 0.1% Tween 20 in dH2O. Recombinant LBP (50 ng/ml) diluted in PBS/0.1% BSA was added to the plates for 1.5 h at room temperature. After washing the plates, biotinylated LPS was added in the presence or absence of cationic peptides. Where indicated, the peptides were either preincubated with biotinylated LPS for 30 min or added to the wells at various times after the addition of biotinylated LPS. In all cases, the plates were washed 1 h after the addition of LPS. Binding of the biotinylated LPS to the immobilized LBP was detected using HRP-conjugated streptavidin diluted 1:2000 in PBS/0.1% BSA. After a 1 h incubation, 3,3',5'5-tetramethylbenzidine (Sigma) was added. The reactions were stopped after 15 min by adding 0.1 ml of 0.18 M sulfuric acid, and the absorbance at 450 nm was determined using an ELISA reader.
TNF- production by RAW 264.7 cells
The murine macrophage cell line RAW 264.7 was obtained from
American Type Culture Collection (Manassas, VA) and grown in DMEM
supplemented with 10% FCS. The cells were plated at
106 cells/well in 24-well plates, incubated
overnight, then stimulated with LPS for 6 h. The culture
supernatants were assayed for TNF- using an ELISA (Endogen, Hornby,
ON) that could detect <50 pg/ml TNF-.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which cationic antimicrobial peptides block the
activation of macrophages by LPS has not been elucidated. We
hypothesized that they block the first step in the process, the
interaction of LPS with LBP. To test this, we used an assay that
measures the binding of biotinylated LPS to immobilized LBP. Fig. 1 shows that amounts of LPS ranging from
10 to 500 ng/ml produced a linear binding curve. Because previous
studies showed that the CP29 cecropin:melittin hybrid cationic
antimicrobial peptide effectively inhibited LPS-induced TNF-
production by RAW 264.7 cells (7), we asked whether this
peptide could block the binding of biotinylated LPS to LBP in this
assay. When 10 µg/ml (3.4 µM) CP29 was added at the same time as
LPS, it substantially reduced the ability of the LPS to bind to LBP
(Fig. 1). The dose-response curve for LPS binding was shifted
10-fold, i.e., in the presence of the CP29 peptide 10 times as much
LPS was required to yield the same amount of LPS bound to LBP compared
with when the peptide was not present. CP29 reduced the binding of LPS
to LBP almost completely when 45 ng/ml LPS was added to the well and by
>80% when 450 ng/ml LPS was added to the well. Thus, the CP29
cationic peptide strongly inhibits the binding of LPS to LBP.
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. In these experiments, we analyzed the
inhibition of the LPS-LBP interaction by the CP29 peptide as well as by
polymyxin B, a cationic antibiotic that causes substantial inhibition
of LPS-induced TNF- production (see Fig. 3B). Fig. 2A shows
that as little as 100 ng/ml polymyxin B (70 nM) or 10 ng/ml CP29 (3.4
nM) could markedly inhibit the binding of LPS to LBP when 45 ng/ml LPS
was added to the wells. The ability of polymyxin B to inhibit the
LPS-LPB interaction leveled off at 60–70% inhibition when 1–10
µg/ml (0.7–7 µM) polymyxin B was added to the wells, whereas
increasing the amount of CP29 from 1 µg/ml (0.34 µM) to 10 µg/ml
(3.4 µM) resulted in greater inhibition, with 10 µg/ml CP29 causing
nearly complete inhibition of the LPS-LBP interaction, as seen also in
Fig. 1
. Despite these differences, it is clear that both CP29 and
polymyxin B are potent inhibitors of the LPS-LBP interaction.
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A). At least 10-fold less CP29 or polymyxin B was required
to block the LPS-LBP interaction when the peptide was mixed with the
LPS for 30 min before adding the LPS to the immobilized LBP-coated
wells. This observation is consistent with our previous findings that
CP29, polymyxin B, and other cationic peptides bind to LPS
(7) and suggests that this interaction prevents LPS from
binding to LBP.
While CP29 and polymyxin B can block the interaction of LPS with LBP,
they cannot effectively disrupt the binding of LPS to LBP once it has
occurred. Fig. 2B shows that when these peptides were added
to the LBP-coated wells 20 min after the LPS, they were no longer able
to substantially reduce the binding of LPS to LBP.
Structurally different cationic antimicrobial peptides inhibit the LPS-LBP interaction
Antimicrobial peptides have a wide variety of secondary structures
(1, 3). For this study, peptides belonging to different
structural groups were tested for their ability to inhibit LPS-LBP
interaction. We tested 1) the human defensins HNP-1 and HBD-2, which
have rigid -sheet structures; 2) indolicidin and the indolicidin
variant, CP11CN, which have an extended structure with -turn
elements (23), 3) Bac 2A-NH2 which
is a linear form of the loop peptide bactenecin (24), 4)
the CP29, CEMA, and CP208 synthetic melittin:cecropin hybrids, which
are amphipathic -helical peptides (2, 7), and 5) the
cyclic bacterial-derived peptide antibiotics polymyxin B and
gramicidin S.
We compared the abilities of cationic peptides belonging to these
different structural groups to inhibit the LPS-LBP interaction (Fig. 3A). At the same time, we compared their abilities to block
LPS-induced TNF- production by the RAW 264.7 macrophage cell line
(Fig. 3B). CP29 and CEMA, synthetic -helical peptides,
were the most effective peptides at blocking both the LPS-LBP
interaction and the production of TNF- by RAW 264.7 cells. They
consistently caused 85–95% inhibition of the LPS-LBP interaction
while reducing LPS-induced TNF- production by >75%. In contrast to
CP29, CP208, an -helical peptide that is related to CP29 but that
lacks the tryptophan found at the N terminus of CP29, had little effect
on the LPS-LBP interaction. Consistent with this observation, this
peptide did not significantly reduce LPS-induced TNF- production. We
have previously shown that CP208 binds LPS poorly compared with CP29
and CEMA and that it has little antimicrobial activity
(7). Thus, the ability of the -helical cationic
peptides to inhibit LPS·LBP interactions depends on structural
features in addition to their positive charges.
Of the other structural groups of cationic peptides, Bac
2A-NH2, gramicidin S, and polymyxin B all caused
significant (55–80%) inhibition of the LPS-LBP interaction (Fig. 3A). For gramicidin S and polymyxin B, this correlated with
their ability to block LPS-induced TNF- production by 60–80% (Fig. 3B). In contrast, Bac 2A-NH2
repeatedly caused only a modest (30%) inhibition of TNF-
production even though it inhibited the LPS-LBP interaction by 75%.
The human neutrophil peptide -defensin HNP-1 and the human
-defensin HBD-2, which belong to the -sheet
class of cationic antimicrobial peptides, both exhibited only a modest
ability (40% inhibition) to block the LPS-LBP interaction, and this
correlated with their modest ability (25–40% inhibition) to block
LPS-induced TNF- production. Indolicidin and the indolicidin variant
CP11CN both significantly inhibited the LPS-LBP interaction (50%
inhibition), but to a lesser extent than CP29. This correlated with
their lower ability to inhibit TNF- production (40% inhibition)
as compared with CP29. Thus, with the exception of Bac
2A-NH2, the ability of different cationic
peptides to inhibit LPS-induced TNF- production correlated well with
their ability to block the binding of LPS to LBP. The strong
correlation (R2 = 0.921) between these two
properties is illustrated in Fig. 3C. Moreover, the nearly
1:1 correlation between the inhibition of LPS-LBP interaction and the
inhibition of TNF- release suggests that the ability of the peptides
to block the LPS-LBP interaction may be a major mechanism by which they
block the ability of LPS to activate macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical peptide CP29 had the best
ability to inhibit both the LPS-LBP interaction and LPS-induced TNF-
secretion by macrophages. It significantly inhibited the LPS-LBP
interaction at concentrations as low as 10 ng/ml (3.4 nM), and 10
µg/ml (3.4 µM) CP29 caused nearly complete inhibition of the
LPS-LBP interaction. The other cationic peptides tested including Bac
2A-NH2, the indolicidins, and gramicidin S also
inhibited both the LPS-LBP interaction and LPS-induced TNF-
production. Bac 2A-NH2 was different from the
other peptides in that it caused moderate inhibition of LPS-LBP
interaction but had only a minor effect on LPS-induced TNF-
production. However, in general, the relative ability of different
peptides to block the binding of LPS to LBP correlated strongly with
their ability to block LPS-induced TNF- production by RAW 264.7
cells. Thus, the ability of cationic peptides to block macrophage
activation by LPS may be due in large part to their ability to block
the binding of LPS to LBP. This would presumably block the transfer of
LPS to CD14 by LBP, greatly decreasing the ability of LPS to activate
macrophages. Le Roy et al. recently showed that mAbs that block either the binding of LPS to LBP or the binding of LBP to CD14 are potent inhibitors of LPS toxicity in vivo and also block LPS-induced TNF production by RAW 264.7 cells (25). Together with our data, these results argue that inhibiting the LPS-LBP interaction is likely to be a very specific and efficient way to reduce or prevent LPS-induced inflammatory responses. The use of cationic peptides, in combination with anti-LBP mAbs, may be a very potent anti-endotoxin treatment. Our results also suggest that LPS-induced production of cationic peptides (26) may limit the magnitude of inflammatory responses by preventing further LPS signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert E. W. Hancock, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address:
3 Abbreviations used in this paper: LBP, LPS binding protein; Bac, bactenecin; HNP-1, human neutrophil peptide-1; HBD-2, human -defensin-2.
Received for publication October 1, 1999. Accepted for publication November 11, 1999.
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References |
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J. D. Schilling, M. A. Mulvey, C. D. Vincent, R. G. Lorenz, and S. J. Hultgren Bacterial Invasion Augments Epithelial Cytokine Responses to Escherichia coli Through a Lipopolysaccharide-Dependent Mechanism J. Immunol., January 15, 2001; 166(2): 1148 - 1155. [Abstract] [Full Text] [PDF]
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A. Risso Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity J. Leukoc. Biol., December 1, 2000; 68(6): 785 - 792. [Abstract] [Full Text]
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O. Levy A Neutrophil-Derived Anti-Infective Molecule: Bactericidal/Permeability-Increasing Protein Antimicrob. Agents Chemother., November 1, 2000; 44(11): 2925 - 2931. [Full Text]
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M. G. Scott, C. M. Rosenberger, M. R. Gold, B. B. Finlay, and R. E. W. Hancock An {alpha}-Helical Cationic Antimicrobial Peptide Selectively Modulates Macrophage Responses to Lipopolysaccharide and Directly Alters Macrophage Gene Expression J. Immunol., September 15, 2000; 165(6): 3358 - 3365. [Abstract] [Full Text] [PDF]
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) or
biotinylated LPS plus 10 µg/ml (3.4 µM) CP29 (
) were added to
wells containing recombinant LBP immobilized using anti-LBP Abs.
Binding of the biotinylated LPS to the immobilized LBP was detected
using HRP-conjugated streptavidin. The values represent the mean and SE
for the averages of duplicate samples from three independent
experiments. Where no error bars are shown, they were smaller than the
symbols.
) for 30 min at
37°C before being added to the immobilized LBP. Alternatively, the
LPS was added to the wells containing the immobilized LBP at the same
time as either CP29 (
), as in Fig. 1