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by Blocking the Binding of LPS to CD14+ Cells1
*
Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan;
Research Division of Innate Immunity, Matsuzono Pharmacy, Iwate, Japan;
Laboratory of Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Science School of Pharmacy, Tokyo, Japan;
Central Research Laboratories, Seikagaku Corporation, Tokyo, Japan; and
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Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois-Lausanne, Lausanne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/
-defensins and cathelicidins) that
contribute to the innate host defense by killing invading
micro-organisms. In this study we evaluated the LPS-neutralizing
activities of cathelicidin peptides human CAP18 (cationic antibacterial
proteins of 18 kDa) and guinea pig CAP11 using the CD14+
murine macrophage cell line RAW264.7 and the murine endotoxin shock
model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited
the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise,
Northern and Western blot analyses indicated that CAP18 and CAP11
suppressed LPS-induced TNF- mRNA and protein expression by RAW264.7
cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities,
and they strongly suppressed the interaction of LPS with LPS binding
protein that mediates the transport of LPS to CD14 to facilitate the
activation of CD14+ cells by LPS. Moreover, when CAP18 and
CAP11 were preincubated with RAW264.7 cells, they bound to the cell
surface CD14 and inhibited the binding of FITC-LPS to the cells.
Furthermore, in the murine endotoxin shock model, CAP18 or CAP11
administration inhibited the binding of LPS to CD14+ cells
(peritoneal macrophages) and suppressed LPS-induced TNF- expression
by these cells. Together these observations indicate that cathelicidin
peptides CAP18 and CAP11 probably exert protective actions against
endotoxin shock by blocking the binding of LPS to CD14+
cells, thereby suppressing the production of cytokines by these cells
via their potent binding activities for LPS and
CD14. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, IL-6, and other cytokines, which mediate the
development of septic shock (1). In certain animal models
TNF- is principal for the lethality induced by LPS
(2, 3, 4). Recent research has revealed the detailed
mechanisms by which LPS activates mononuclear phagocytes
(5, 6, 7, 8). On release, LPS interacts with the LPS-binding
protein (LBP),3 an
acute phase reactant that is present in the blood and transfers LPS to
CD14, the primary receptor for LPS that exists as a soluble form in
blood and as a GPI-linked molecule on the surface of mononuclear
phagocytes (9, 10, 11). LPS-CD14 complexes initiate the
intracellular signaling by binding to the recently defined membrane
proteins Toll-like receptors (TLRs), which are structurally related to
the Drosophila protein Toll and are expressed on mononuclear
phagocytic cells and other cells (5, 6, 7, 8). LPS-CD14
complexes activate NF-
B transcription factor as well as
extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38
mitogen-activated protein kinase, which mediate the production of
inflammatory cytokines (5, 6, 7, 8).
In mammals a number of cationic antibacterial peptides, such as
defensins, are found in blood, secretions, epithelial tissues, and
neutrophil granules (12, 13, 14, 15, 16). They are evolutionary
ancient components that can kill the invading micro-organisms by
perturbing their membranes and contribute to the innate host defense.
Among these peptides, cathelicidin is a novel family of antibacterial
peptides that have been isolated from epithelial tissues and myeloid
cells of human and animal species (17). Precursors of
cathelicidins are characterized by the highly conserved cathelin-like
pro-sequences and variable carboxyl-terminal sequences that correspond
to the mature antibacterial peptides. We have isolated two members of
cathelicidins, CAP18 (cationic antibacterial proteins of 18 kDa) and
CAP11 (cationic antibacterial polypeptide of 11 kDa) from rabbit and
human neutrophils (18, 19), and guinea pig neutrophils
(20, 21), respectively. CAP18 is a precursor form of
cathelicidins, and its carboxyl-terminal antibacterial peptide
comprising 37 aa residues
(L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES37)
has been recently identified in humans (13). In contrast,
CAP11 is a carboxyl-terminal antibacterial peptide cleaved from its
precursor and is a homodimer of
G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43)
joined with one disulfide bond (20, 21). CAP18 and CAP11
peptides exhibit potent antibacterial activities against both
Gram-negative and Gram-positive bacteria (18, 19, 20, 21).
Moreover, it has been found that a CAP18-derived peptide can block
LPS-induced TNF- release and reduce the mortality associated with
endotoxemia in the galactosamine-sensitized murine model
(22). Thus, derivatives of CAP18 could have therapeutic
potential for Gram-negative bacterial sepsis and septic shock
(23). However, little is known about the mechanisms by
which CAP18-derived peptides block the biological activities of LPS.
Moreover, it is unclear whether CAP11, a homologue of CAP18, also has
LPS-neutralizing activity.
In this study, therefore, to elucidate the mechanisms of the protective
actions of CAP18 and the actions of CAP11 on LPS, we investigated the
effects of human CAP18 and guinea pig CAP11 peptides on the binding of
LPS to CD14+ cells and TFN- expression by
these cells using the murine macrophage cell line RAW264.7 and the
murine endotoxin shock model. The results obtained indicated that CAP18
and CAP11 peptides possess LPS- and CD14-binding activities and
exert the protective actions in murine endotoxin shock possibly by
blocking the binding of LPS to CD14+ cells
and suppressing cytokine production by these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC-conjugated LPS (from Escherichia coli 0111:B4),
LPS (from E. coli 0111:B4), 3,3',5,5'-tetramethyl-benzidine
(TMB) liquid substrate system, and
D-galactosamine were purchased from Sigma (St.
Louis, MO). In some experiments E. coli 0111:B4 LPS was
biotinylated with biotin-LC-hydrazide, based on the manufacturer’s
protocol (Pierce, Rockford, IL). Synthetic lipid A LA-15-PP (506) was
obtained from Daiichi Pure Chemicals (Tokyo, Japan). A 37-mer peptide
of human CAP18
(L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRMLVPRTES37),
a guinea pig CAP11 peptide with a free sulfhydryl group of cysteine
residue
(G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43),
and a 20-mer peptide of p40-phox, an NADPH oxidase component
(L1HITQQDNYSVYNTTPSATQ20)
(24) were synthesized by the solid phase method on a
peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan) by F-moc
chemistry. The peptides were eluted from the resin using a standard
protocol and purified to homogeneity by reversed phase HPLC on a
Cosmosil 5C18 column (Nacalai Tesqu, Kyoto,
Japan), using a 0–70% acetonitrile gradient in 0.1% trifluoroacetic
acid. The molecular masses of synthesized peptides were confirmed on a
mass spectrometer (model TSQ 700; Thermo Quest Finnigan, Manchester,
U.K.). To make a dimer form of CAP11, synthesized 43-mer peptide was
oxidized in room air. SDS-urea-PAGE analysis revealed that >95% of
CAP11 peptide was jointed by disulfide bonding. Human neutrophil
peptide-1, human -defensin (hBD)-1, and hBD-2 were purchased from
Peptide Institute (Osaka, Japan). Tissue culture supplies were obtained
from Iwaki Glass (Tokyo, Japan).
Antibodies
As anti-LBP Abs, mouse anti-human LBP mAb 6G3 (HyCult Biotechnology, Uden, The Netherlands) and rat anti-mouse LBP mAb clone 39 (class 2) (25) were used. These anti-LBP mAbs can recognize both free LBP and LBP-LPS complexes, and inhibit the binding of LBP-LPS complexes to CD14. As anti-CD14 Abs, FITC-conjugated rat anti-mouse CD14 mAb rmC5-3 (BD PharMingen, San Diego, CA) and FITC-conjugated rat anti-mouse CD14 mAb 4C1 (26) were used. The 4C1 epitope is distinct from the rmC5-3 epitope (aa residues 308–322, the C-terminal moiety of murine CD14). 4C1 can block the binding of LPS to CD14+ cells, whereas rmC5-3 has little effect on LPS binding (26). Ascites of anti-human CD14 mAb MEM-18, which can recognize an epitope in the region spanning aa 57–64 of human CD14 and block the binding of LPS to CD14 (27), was obtained from HyCult Biotechnology. Rabbit control IgG was prepared from nonimmunized rabbit serum by affinity chromatography using a Hitrap protein A column (Amersham Pharmacia Biotech, Little Chalfont, U.K.).
Cells
Murine macrophage cell line RAW264.7 was obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS (Sanko Junyaku, Tokyo, Japan) at 37°C in 5% CO2. Confluent RAW264.7 cells were detached by washing with 0.05% EDTA in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4).
Assay for the binding of FITC-conjugated LPS to RAW264.7 cells
RAW264.7 cells (5 x 105 cells/ml) were incubated with FITC-conjugated LPS (100 ng/ml) in the absence or the presence of cathelicidin peptide (CAP18 or CAP11, 0.01–10 µg/ml) in RPMI 1640 containing 10% FBS for 15 min at 37°C. After washing the cells with PBS, the binding of FITC-LPS was analyzed by flow cytometry (FACScan; BD Biosciences, Rutherford, NJ), and median fluorescence intensity was determined. Alternatively, RAW264.7 cells were incubated with FITC-conjugated LPS in the presence of anti-human LBP mAb 6G3 (5 µg/ml), which can cross-react with bovine LBP, in RPMI 1640 containing 10% FBS, and the binding of FITC-LPS was analyzed as described above. In some experiments RAW264.7 cells were preincubated with CAP18 or CAP11 peptide (0.1–10 µg/ml) in RPMI 1640 containing 10% FBS for 10 min at 37°C. After washing, the cells were incubated with FITC-LPS (100 ng/ml) in RPMI 1640 containing 10% FBS for 15 min at 37°C, and LPS binding was evaluated by flow cytometry. Moreover, the binding of FITC-LPS was investigated using RPMI 1640 containing 10% mouse serum. Anti-mouse LBP mAb clone 39 suppressed binding of FITC-LPC to RAW264.7 cells in the medium containing 10% mouse serum, and CAP18 and CAP11 peptides also inhibited LPS binding, as in the medium containing 10% FBS (data not shown).
Evaluation of the expression of TNF-
RAW264.7 cells (106 cells/well in a
24-well microplate) were incubated with LPS (100 ng/ml) in the absence
or the presence of cathelicidin peptides (CAP18 or CAP11, 10 µg/ml)
in 500 µl of RPMI 1640 containing 10% FBS for 4 h at 37°C.
After incubation the cells were detached by washing with 0.05%
EDTA/PBS, and the expression of TNF- mRNA and protein was analyzed
by Northern and Western blotting, respectively. In brief, total
cellular RNA (2.5 µg) isolated by the acid guanidinium
thiocyanate-phenol-chloroform extraction method was separated by
electrophoresis on 1% agarose-formaldehyde gel and then transferred
onto nylon membranes (Hybond N+; Amersham
Pharmacia Biotech). RNA was cross-linked with a Funa-UV Linker
(Funakoshi, Tokyo, Japan), and the blots were hybridized with cDNA
probes, which were labeled with a digoxigenin-high prime DNA labeling
kit (Roche, Mannheim, Germany). Probes used were the 0.39-kb TNF-
cDNA (encompassing nucleotides 427–819) (28) obtained by
the PCR amplification of LPS-stimulated RAW264.7 cell cDNA, and the
2.1-kb -actin cDNA (pHFA-1; provided by P. Gunning and L. Kedes,
Stanford University, Palo Alto, CA) (29). In Western blot
analysis the cells suspended in PBS containing 1 mM di-isopropyl
fluorophosphate were disrupted on ice by sonication, and aliquots
(3 x 105 cells for TNF- and 3 x
104 cells for -actin) were subjected to
SDS-PAGE on a 7.5–20% linear gradient of polyacrylamide under
reducing conditions. The resolved proteins were electrotransferred to
Immobilon-P membrane (Millipore, Bedford, MA) using a Trans-Blot SD
apparatus (Bio-Rad, Hercules, CA). The blots were blocked in Block Ace
(Dainippon Pharmaceutical, Osaka, Japan) and probed with rabbit
anti-mouse TNF- Ab (Genzyme, Cambridge, MA) or
anti--actin mAb (Sigma). The blots were further probed with
HRP-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC)
or goat anti-mouse IgG/IgM (Chemicon, Temecula, CA), and proteins
were finally detected with SuperSignal West Pico chemiluminescent
substrate (Pierce). In some experiments RAW264.7 cells were incubated
with LPS in the medium containing 10% mouse serum, and the effect of
cathelicidin peptides on the LPS-induced TNF- expression was
investigated as described above.
Measurement of the LPS-binding activities of cathelicidin peptides
The 96-well microtiter plates (Immulon 2H; Dynex Technologies, Ashford, U.K.) were coated with LPS (100 ng/well) by incubating 50 µl/well of 2 µg/ml LPS in 0.1 M Na2CO3 and 20 mM EDTA, pH 9.6, for 3 h at 37°C. The LPS solution was flicked out, and the plates were rinsed thoroughly under running water and air-dried overnight (30). Excess binding sites were blocked with 100 µl/well PBS containing 1% BSA, and CAP18 or CAP11 peptide (0.02–0.1 µg/well) was incubated for 1 h at 37°C in 50 µl of RPMI 1640 without phenol red (Life Technologies, Grand Island, NY). Alternatively, cathelicidin peptide (0.1 µg/well) was incubated in the presence of LPS (0.5–2.5 µg/well) in 50 µl of RPMI 1640. After washing, affinity-purified rabbit anti-CAP18 or anti-CAP11 Ab (50 µl/well; 2 µg/ml in PBS containing 0.1% BSA) was added and incubated for 1 h at 37°C. The Ab solution was then rinsed out, and HRP-conjugated goat anti-rabbit IgG (50 µl/well; diluted 2000-fold in PBS containing 0.1% BSA) was incubated in the wells for 1 h at room temperature. Finally, TMB liquid substrate (100 µl/well) was incubated until sufficient color had developed (5–15 min). The reaction was stopped by adding 100 µl/well 0.18 M sulfuric acid, and the absorbance at 450 and 560 nm was quantitated in a microtiter plate reader. Anti-CAP18 and anti-CAP11 sera were raised in rabbits with the use of synthetic CAP18 and CAP11 peptides covalently coupled to keyhole limpet hemocyanin, respectively, and Abs were purified by affinity chromatography using the synthetic peptide-conjugated, epoxy-activated, Sepharose (Amersham Pharmacia Biotech).
In addition, the LPS-binding activities of CAP18 and CAP11 peptides were quantified by hemagglutination assay using LPS-sensitized erythrocytes (19). For sensitization, 1% sheep erythrocyte suspension (10 ml; Toyo Bio, Tokyo, Japan) was incubated with 2 ml of 100 µg/ml LPS in saline for 30 min at 37°C. After washing and suspended in PBS, 1% suspension of LPS-sensitized erythrocytes (50 µl) was mixed with a 2-fold serial dilution of cathelicidin peptide (50 µl; 0.31–10 µg/ml in PBS) in the absence or presence of LPS or lipid A (0.06–2.0 µg/ml) in a U-bottom microtiter plate and incubated for 1 h at 37°C. The LPS-binding activity of CAP18 or CAP11 peptide was expressed as a minimum agglutinating concentration (MAC), whereas the inhibition of CAP18- or CAP11-mediated hemagglutination by LPS or lipid A was expressed as a minimum inhibitory concentration (MIC). In some experiments sheep erythrocytes were sensitized with lipid A by incubating with 25 µg/ml lipid A in saline for 30 min at 37°C. After washing, hemagglutination was assayed in saline.
Assay for the interaction of LPS with LBP
LPS (100 ng/well) was immobilized to the 96-well microtiter plates as described above. After blocking, RPMI 1640 containing 0.1, 1, or 10% FBS (50 µl/well) was added and incubated for 1 h at 37°C. The plates were then washed, and 50 µl/well anti-LBP mAb 6G3 (25 nM in PBS containing 0.1% BSA) was incubated in the wells for 1 h at 37°C. The mAb solution was rinsed out and replaced with 50 µl/well HRP-conjugated rabbit anti-mouse IgG (diluted 1000-fold in PBS containing 0.1% BSA; DAKO, Glostrup, Denmark) for 1 h at room temperature. Finally, the binding of LBP to the immobilized LPS was detected by incubating with TMB liquid substrate (100 µl/well). Alternatively, the microtiter plates were preincubated with CAP18 or CAP11 peptide (0.5–2.5 µg/well) in 50 µl/well RPMI 1640 for 1 h at 37°C. After washing, RPMI 1640 containing 10% FBS (50 µl/well) was added, and the binding of LBP was determined as described above. In some experiments the binding of mouse LBP to the immobilized LPS was assessed using mouse serum, rat anti-mouse LBP mAb clone 39, and HRP-conjugated rabbit anti-rat IgG (DAKO).
Assay for the binding of LBP to cathelicidin peptides
The microtiter plates were coated with cathelicidin peptides (2.5 µg/well) by incubating 50 µl/well of 50 µg/ml CAP18 or CAP11 in PBS overnight at room temperature. After blocking, RPMI 1640 containing 10% FBS (50 µl/well) was added and incubated for 1 h at 37°C. The plates were then washed, and 50 µl/well anti-LBP mAb 6G3 (25 nM in PBS containing 0.1% BSA) was incubated in the wells for 1 h at 37°C. The mAb solution was rinsed out and replaced with 50 µl/well HRP-conjugated rabbit anti-mouse IgG (diluted 1000-fold in PBS containing 0.1% BSA) for 1 h at room temperature. Finally, the binding of LBP to the immobilized cathelicidin peptides was detected by TMB reaction. As a positive control, biotinylated LPS (100 ng/well) was added to the CAP18- or CAP11-immobilized plates and incubated for 1 h at 37°C in 50 µl/well RPMI 1640. The LPS solution was then rinsed out, and 50 µl/well HRP-conjugated streptavidin (diluted 5000-fold in PBS containing 0.1% BSA; DAKO) was incubated for 1 h at 37°C. The binding of biotinylated LPS to the immobilized CAP18 or CAP11 was finally detected by TMB reaction.
Flow cytometric assay for the expression of CD14 and the binding of cathelicidin peptides
To analyze the effect of cathelicidin peptides on CD14 expression, RAW264.7 cells (5 x 105 cells/ml) were incubated without or with cathelicidin peptides (CAP18 or CAP11, 10 µg/ml) or LPS (100 ng/ml) in RPMI 1640 containing 10% FBS for 15 min at 37°C and further incubated with FITC-conjugated rat anti-mouse CD14 mAb rmC5-3 (2.5 µg/ml) or FITC-conjugated rabbit anti-mouse IgG (DAKO), as a negative control, for 15 min at 37°C. After washing, the binding of anti-CD mAb was measured by flow cytometry.
To evaluate the binding of cathelicidin peptides to the CD14+ cells, RAW264.7 cells (5 x 105 cells/ml) were incubated with CAP18 or CAP11 (10 µg/ml) in the absence or the presence of CAP11 or CAP18 (20 µg/ml), respectively, in RPMI 1640 containing 10% FBS for 15 min at 37°C. The cells were then washed and incubated with 2 µg/ml rabbit anti-CAP18 or anti-CAP11 Ab or rabbit control IgG, as a negative control, in RPMI 1640 containing 10% FBS for 15 min at 37°C. After washing, the cells were further incubated with FITC-conjugated goat anti-rabbit IgG (diluted 1000-fold in RPMI 1640 containing 10% FBS; Organon Teknika), and the binding of cathelicidin peptides was analyzed by flow cytometry. RAW264.7 cells were also incubated with p40-phox peptide (10 µg/ml), a control peptide, and then the peptide binding was examined using affinity-purified rabbit anti-p40-phox Ab (24) and FITC-conjugated goat anti-rabbit IgG by flow cytometry.
Furthermore, the binding of cathelicidin peptides to CD14 was examined using neutralizing anti-mouse CD14 mAb 4C1 that can recognize the murine CD14 epitope and inhibit the binding of LPS to CD14 (26). RAW264.7 cells (5 x 105 cells/ml) were preincubated without or with cathelicidin peptides (CAP18 or CAP11, 10 µg/ml) or LPS (100 ng/ml), as a positive control, for 15 min at 37°C in RPMI 1640 containing 10% FBS. The cells were then added with 50 ng/ml FITC-conjugated anti-CD14 mAb 4C1 or FITC-conjugated rabbit anti-mouse IgG, as a negative control, and further incubated for 15 min at 37°C. After washing, the binding of FITC-conjugated anti-CD14 mAb 4C1 was analyzed by flow cytometry. Moreover, the binding of FITC-conjugated anti-CD14 mAb 4C1 to RAW264.7 cells was investigated using RPMI 1640 containing 10% mouse serum.
Evaluation of the effect of cathelicidin peptides on murine endotoxin shock model
To determine the protective effect of cathelicidin peptides
against the lethal activity of LPS, we used
D-galactosamine-sensitized mice that are highly susceptible
to LPS (31). Male C57BL/6 mice, aged 10 wk, were purchased
from Japan SLC (Shizuoka, Japan). D-galactosamine (18
mg/0.3 ml of saline), FITC-conjugated LPS (100 ng/0.2 ml of saline),
and cathelicidin peptides (CAP18 or CAP11, 10 µg/0.2 ml of saline)
were sequentially injected i.p. into mice, and deaths were recorded
every 24 h until day 6 after the injection. In some experiments,
75 min after LPS challenge mice were sacrificed by drawing blood from
the heart, and sera were prepared. Concurrently, peritoneal fluids were
harvested by washing the peritoneal cavities with 5 ml of PBS, and the
supernatants and pelleted cells (peritoneal macrophages) were
recovered. Serum TNF- levels were determined using a commercially
available mouse TNF- ELISA kit (Endogen, Woburn, MA) that can detect
<50 pg/ml TNF-. Furthermore, using peritoneal macrophages, the
binding of FITC-conjugated LPS was analyzed by flow cytometry, and the
expression of TNF- was investigated by Northern blotting and Western
blotting, as described in the above sections. In addition, the LBP
levels in sera and peritoneal supernatants were quantitated by Western
blot analysis using rat anti-mouse LBP mAb clone 39 and HRP-conjugated
rabbit anti-rat IgG. To measure the relative amounts, the detected
65-kDa LBP bands were quantified using a scanning densitometer
(MasterScan System; Scanalytics, Fairfax, VA), and LBP levels in the
peritoneal supernatants were corrected based on the volume of
peritoneal fluids recovered.
Statistical analysis
Data are shown as the mean ± SD. Statistical significance was determined by one-way ANOVA with multiple comparison test (StatView; Abacus Concepts, Berkeley, CA), and results were considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the effects of cathelicidin peptides (CAP18 and
CAP11) on the binding of FITC-conjugated LPS to
CD14+ cells by flow cytometry using the murine
macrophage cell line RAW264.7. When FITC-LPS was incubated with
RAW264.7 cells, it bound to the cells serum-dependently (Fig. 1
, upper panel); FITC-LPS was
hardly bound to the cells in the absence of serum, and the fluorescence
intensity was almost the same as that of background without FITC-LPS.
Furthermore, FITC-LPS was incubated with RAW264.7 cell in the presence
of anti-LBP mAb (5 µg/ml) that can recognize both free LBP and
LBP-LPS complexes and inhibit the binding of LBP-LPS complexes to CD14.
The anti-LBP mAb inhibited the binding of FITC-LPS to RAW264.7
cells by 96.0 ± 3.5% (n = 3), indicating that
the LPS binding requires serum LBP.
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, lower
panel). Their effects were dose dependent (Fig. 2), and CAP11 (IC50
= 0.05 µg/ml or 0.005 µM) was more potent than CAP18
(IC50 = 0.25 µg/ml or 0.056 µM) in inhibiting
the binding of FITC-LPS to RAW264.7 cells. Furthermore, the effect of
CAP18 and CAP11 on the TNF- expression was examined by Northern and
Western blot analyses (Fig. 3). CAP18 and
CAP11 (10 µg/ml each) completely suppressed LPS-induced TNF-
expression by RAW264.7 cells at both mRNA and protein levels.
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To clarify the mechanisms by which CAP18 and CAP11 inhibit the
binding of LPS to CD14+ cells, we investigated
the LPS-binding activities of CAP18 and CAP11 using LPS-immobilized
microtiter plates. CAP18 and CAP11 bound to the LPS-immobilized plates
in a dose-dependent fashion (Fig. 4A), and the binding was
dose-dependently inhibited by LPS added to the plates (Fig. 4B). Noticeably, the binding of CAP11 was more potently
inhibited by LPS (IC50 = 0.07 µg LPS/well)
compared with that of CAP18 (IC50 = 0.28 µg
LPS/well).
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Using the LPS-immobilized plates, we next examined the effects of CAP18
and CAP11 on the binding of LPS to LBP that catalyzes the transfer of
LPS to CD14. When the LPS plates were incubated with FBS, serum LBP
bound to the LPS plates dose-dependently on the concentrations of serum
used (Fig. 5A). Moreover, the
LPS plates were pretreated with CAP18 or CAP11, and then the LPS-LBP
interaction was analyzed. The binding of LBP to the LPS plates was
inhibited in a dose-dependent manner by CAP18 and CAP11 added to the
plates (Fig. 5B). Importantly, CAP11
(IC50 = 0.06 µg/well or 5.7 pmol/well) was more
potent than CAP18 (IC50 = 0.2 µg/well or 44.5
pmol/well) in inhibiting LPS-LBP binding.
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These above observations indicate that both CAP18 and CAP11 can bind to LPS, but not LBP, thereby inhibiting the interaction of LPS with LBP. Moreover, CAP11 has more potent activities than CAP18 for binding to LPS and inhibiting LPS/LBP interaction.
Effect of CAP18 and CAP11 on CD14 expression, and binding of CAP18 and CAP11 to CD14+ cells
It is possible that cathelicidin peptides may alter CD14
expression, thereby affecting the binding of LPS to
CD14+ cells. To check this, we investigated the
expression of CD14 after treatment of RAW264.7 cells with CAP18 or
CAP11 by flow cytometry using FITC-conjugated anti-mouse CD14 mAb
rmC5-3. Neither CAP18, CAP11 (10 µg/ml each), nor LPS (100 ng/ml)
changed the CD14 expression on RAW264.7 cells (Fig. 6).
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). The inhibition was dose dependent on
the peptides used, and CAP11 (IC50 = 1.4 µg/ml
or 0.133 µM) was more potent than CAP18 (IC50 =
6 µg/ml or 1.362 µM) in inhibiting the binding of FITC-LPS to
RAW264.7 cells. These observations indicate that CAP18 and CAP11 can
bind to the cells and affect LPS binding. In fact, flow cytometric
assay using anti-CAP18 and anti-CAP11 Abs revealed that CAP18
and CAP11 could bind to RAW264.7 cells (Fig. 8A). On the contrary, p40-phox
peptide (10 µg/ml), a control peptide, did not essentially bind to
the cells (data not shown). Of interest, CAP11 (20 µg/ml) inhibited
the binding of CAP18 (10 µg/ml) to RAW264.7 cells by 72.1 ±
5.1% (n = 3). Conversely, CAP18 (20 µg/ml) inhibited
the binding of CAP11 (10 µg/ml) to the cells by 72.2 ± 14.1%
(n = 3). These observations indicate that CAP18 and
CAP11 specifically bind to RAW264.7 cells, and the two peptides may
share the same receptors (binding sites) on the cells.
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B),
suggesting that the epitope, which can be recognized by mAb 4C1, is
located on murine CD14 next to the LPS binding site. Next, RAW264.7
cells were preincubated with CAP18 or CAP11 (10 µg/ml each), and the
binding of FITC-conjugated anti-CD14 mAb 4C1 to the cells was
determined. Similar to the results with LPS, preincubation with CAP18
or CAP11 inhibited the binding of FITC-conjugated anti-CD14 mAb 4C1
to RAW264.7 cells by 43.0 ± 10.6% (n = 4) and
56.8 ± 10.3% (n = 3), respectively (Fig. 8C). These observations suggest that CAP18 and CAP11 can
bind to CD14 near the LPS binding site and influence the binding of LPS
to CD14+ cells.
In separate experiments we confirmed using 10% mouse serum-containing
medium that CAP18 and CAP11 could suppress the binding of
FITC-conjugated LPS to RAW264.7 cells, LPS-induced TNF- expression
by RAW264.7 cells, binding of LBP to LPS, and binding of
FITC-conjugated anti-CD14 mAb 4C1 to RAW264.7 cells, as observed
with 10% FBS-containing medium (data not shown). These observations
indicate that CAP18 and CAP11 could function in mice.
Effects of CAP18 and CAP11 on the murine endotoxin shock model
Using D-galactosamine-sensitized mice, we assessed the
actions of cathelicidin peptides on the lethal activity of LPS in vivo
(31). D-galactosamine administration
sensitized mice to the lethal effect of LPS, and 90% of the sensitized
mice died within 24 h after the i.p. injection of 100 ng of LPS
(Fig. 9A). Noticeably, the
administration of CAP18 or CAP11 (10 µg/mouse) increased the survival
rate to 80 and 100%, respectively. Moreover, CAP18 and CAP11
administration markedly lowered the LPS-induced increase in serum
TNF- levels (p < 0.05; Fig. 9B). In
addition, the effect of cathelicidin peptides on the binding of
FITC-conjugated LPS to peritoneal macrophages
(CD14+ cells) was analyzed by flow cytometry.
CAP18 or CAP11 administration significantly suppressed the binding of
FITC-LPS to peritoneal macrophages (p < 0.001;
Fig. 10). Concurrently, TNF-
expression was investigated using peritoneal macrophages. Northern and
Western blot analyses indicated that CAP18 or CAP11 administration
markedly suppressed LPS-induced TNF- mRNA and protein expression by
peritoneal macrophages (Fig. 11).
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by these cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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appears to be very important for the development of endotoxin shock
(2, 3, 4). Endotoxin shock could occur in the process of
antibiotic therapy for the underlying bacterial infections in septic
syndrome. In this context, some antibiotics are known to kill the
bacteria but stimulate the release of LPS from the outer membrane of
the dying bacteria, thereby provoking the occurrence of endotoxin shock
(33, 34); they cannot neutralize the biological activity
of released LPS. Thus, a drug that suppresses the actions of LPS could
be a reasonable additional therapy for endotoxin shock or sepsis caused
by Gram-negative bacterial infections. Until now, several strategies,
including neutralizing Abs against LPS, LBP, or cytokines, have been
tested to prevent the cascade of LPS-induced inflammatory reactions
(2, 25, 35, 36, 37). Recently, however, much attention has
focused on the low m.w. cationic antibacterial peptides that possess
both the antibacterial and LPS-neutralizing activities. Originally,
those peptides are known to function in the innate host defense against
microbial infections by impairing the membranes of targeted organisms
(12, 13, 14, 15). Scott et al. (38, 39) synthesized the hybrid peptides derived from silk moth cecropin and bee melittin peptides and examined their effects on the binding of LPS to LBP. Furthermore, Iwagaki et al. (40) investigated the binding of LPS to CD14+ cells using synthetic antiendotoxin peptides that were designed to mimic the structures of polymyxin B, which inhibits the biological activities of LPS through its high affinity binding to the lipid A moiety of LPS. In addition, Dankesreiter et al. (41) synthesized the hybrid peptides containing LPS-binding domains from Limulus anti-LPS factor and LBP and examined their effects on the interaction of LPS with CD14+ cells. However, the detailed mechanisms by which those synthetic peptides exert the LPS-neutralizing activities are not fully understood, and it is unclear whether those synthetic peptides could influence the CD14 (LPS receptor) expression and/or bind to CD14 to affect the LPS binding. Furthermore, the information is very limited about the LPS-neutralizing activities of naturally occurring antibacterial peptides in mammals.
Recently, it has been reported that a peptide derived from human CAP18,
a member of cathelicidins, can lower serum TNF- levels and protect
mice from lethal endotoxin shock (22). In this study to
clarify the possible mechanisms for the protective actions of
cathelicidin peptides, we evaluated the LPS-neutralizing activities of
human CAP18 and guinea pig CAP11 peptides using the
CD14+ murine macrophage cell line RAW264.7 and
the murine endotoxin shock model. Flow cytometric analysis revealed
that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to
RAW264.7 cells. Likewise, Northern and Western blot analyses indicated
that CAP18 and CAP11 suppressed LPS-induced TNF- mRNA and protein
expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed
LPS- or lipid A-binding activities, and they strongly suppressed the
interaction of LPS with LBP that transports LPS to CD14. Moreover,
CAP18 and CAP11 could bind to CD14 and inhibited the binding of
FITC-LPS to the CD14+ cells, although they did
not alter CD14 expression on the cells. Furthermore, CAP18 and CAP11
administration inhibited LPS binding to CD14+
cells (peritoneal macrophages) and suppressed LPS-induced TNF-
expression by these cells in murine endotoxin shock model. Among TLRs,
TLR4 is reported to be required for the LPS-CD14 complex to initiate
intracellular signaling and induce inflammatory responses (42, 43). Together these observations indicate that cathelicidin
peptides CAP18 and CAP11 could exert the protective actions in murine
endotoxin shock by blocking the LBP-mediated transport of LPS to
CD14+ cells, thereby possibly suppressing the
TLR4-mediated cytokine (such as TNF-) production by these cells via
their potent binding activities for LPS and CD14.
Previously, the peptides derived from human and rabbit CAP18, members
of the cathelicidin family, were shown to have LPS-binding activities
(18, 19). In this study we have revealed that a 37-mer
peptide of human CAP18 and CAP11 (a guinea pig homologue of CAP18)
possesses LPS- or lipid A-binding activities and can suppress the
biological actions of LPS. Thus, among cathelicidin peptides, only
CP18-derived peptides and CAP11 are shown to have LPS-binding and
neutralizing activities. LPS and lipid A, which entirely contains the
endotoxic activities of LPS, are amphipathic molecules with negative
charges, and their negatively charged groups (phosphate groups and
carboxyl groups of 2-keto-3-deoxyoctulosonic acid, Kdo) are important
for the expression of biological activities (32).
Interestingly, the potential LPS binding domain of LPS-neutralizing
proteins such as Limulus anti-LPS factor, LBP, and
bactericidal/permeability-increasing protein contains a positively
charged amphipathic structure, and the positively charged groups of the
LPS-binding domain are thought to interact with the negatively charged
groups of lipid A and LPS (44). Consistent with this,
CAP18 and CAP11 peptides display the amphipathic helical structure
with hydrophilic (positively charged) and hydrophobic surfaces
(20). Thus, the structural feature of CAP18 and CAP11
peptides (positively charged amphipathic structure) is considered
important for their LPS-binding capacities. Importantly, CAP11
exhibited more potent LPS-neutralizing activities than CAP18, and pI
values for CAP18 and CAP11 were 11.12 and 12.31, respectively
(Genetyx-Mac computer system; Software Development, Tokyo, Japan).
These observations indicate that the cationicities of antibacterial
peptides may correlate with their LPS-binding activities. Supporting
this, we have demonstrated that human -defensin (human neutrophil
peptide-1) and -defensins (hBD-1 and hBD-2) with pI values of 8.28,
8.55, and 9.25, respectively, have much lower LPS-binding activities
than CAP18 and CAP11, as assessed by LPS-sensitized hemagglutination
assay, and they can have little inhibitory effect on the binding of
FITC-conjugated LPS to RAW264.7 cells or the suppression of LPS-induced
TNF- expression by these cells (data not shown). However, in
contrast to the amphipathic helical structure of CAP18 and CAP11
peptides, defensins exhibit the positively charged amphipathic
-sheet structure (12, 14, 15), suggesting that these
structures may be also involved in the difference between
LPS-neutralizing activities of defensins and cathelicidin peptides
(CAP18 and CAP11).
Using a neutralizing anti-mouse CD14 mAb 4C1, we have indicated
that CAP18 and CAP11 can bind to murine CD14 and inhibit the binding of
LPS to CD14+ cells. However, neither an epitope
for 4C1 nor an LPS binding site has been identified in murine CD14
(26). In contrast, it has been demonstrated that LPS can
bind to the negatively charged domain in human CD14 spanning aa 57–64
(D57ADPRQYA64), which can
be recognized by a neutralizing anti-human CD14 mAb MEM-18
(27). Moreover, it has been reported that the region at
position 9–13 (D9DEDF13)
comprising anionic amino acids is also involved in the binding of LPS
to human CD14 (45). In separate experiments we
investigated the effect of CAP18 and CAP11 on the binding of
anti-CD14 mAb MEM-18 to CD14 using CD14-expressing U937 cells that
had been differentiated by incubation with 10 nM PMA for 2 days
(46). Importantly, CAP18 and CAP11 (10 µg/ml) inhibited
the binding of MEM-18 (1/20 dilution of ascites) to
CD14+ U937 cells by 50–80% (data not shown),
indicating that CAP18 and CAP11 are able to bind to the LPS binding
site on human CD14. Thus, it is tempting to speculate that CAP18 and
CAP11 can bind to the negatively charged LPS-binding domain(s), if any,
present in murine CD14, thereby inhibiting the binding of LPS to mouse
CD14+ cells. Supporting this, the amino acid
sequence homology search (Genetyx-Mac computer system) has revealed
that murine CD14 contains two negatively charged regions,
D11EESC15 and
E57ADLGQFT64, corresponding
to the D9DEDF13 and
D57ADPRQYA64 sequences of
human CD14, respectively (47). However, it has not been
determined whether the two regions of murine CD14 are involved in LPS
binding. Moreover, CAP18 and CAP11 peptides could only partially (at
most 
50%) inhibit the binding of anti-CD14 mAb 4C1 to
CD14+ RAW264.7 cells, and the exact binding
site(s) for CAP18 and CAP11 in murine CD14 has not been defined. Thus,
the mechanism by which the cell surface-bound peptides suppress the
binding of LPS to CD14 molecule remains speculative and needs further
experimental development in the future.
Human CAP18 and guinea pig CAP11 peptides can exhibit the antibacterial activities against Gram-negative and Gram-positive bacteria in the extracellular milieu containing a physiological concentration of NaCl (150 mM), whereas the antibacterial activities of defensins are completely lost under these conditions (48). Moreover, we have argued in this study that CAP18 and CAP11 could bind to LPS and CD14 and neutralize the activities of LPS. To suppress the cascade of LPS-induced inflammatory reactions, several agents, including neutralizing Abs against LPS, LBP, or cytokines, have been examined (2, 25, 35, 36, 37). Unlike these agents, CAP18 and CAP11 would be expected not only to block the onset of LPS-triggered inflammatory reactions by binding directly to LPS and CD14, but also to overcome the underlying Gram-negative bacterial infections in the septic shock syndrome. Thus, CAP18, CAP11, and their derivatives could be attractive candidates for adjunctive therapy in Gram-negative bacterial sepsis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan., E-mail address: nagaokai{at}med.juntendo.ac.jp
3 Abbreviations used in this paper: LBP, LPS-binding protein; CAP18, cationic antibacterial protein of 18 kDa; CAP11, cationic antibacterial polypeptide of 11 kDa; TLR, Toll-like receptor; TMB, 3,3',5,5'-tetramethyl-benzidine; hBD, human -defensin; MAC, minimum agglutinating concentration; MIC, minimum inhibitory concentration.
Received for publication April 3, 2001. Accepted for publication July 9, 2001.
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