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J. Biol. Chem., Vol. 277, Issue 27, 24315-24320, July 5, 2002
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From the Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan
Received for publication, July 25, 2001, and in revised form, April 10, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Toll-like receptor 2 (TLR2) has been recognized
to mediate cell signaling in response to peptidoglycan (PGN), a major
cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of
recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct
interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN
(iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast,
sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN
cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been
incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced
NF- The innate immune system plays key roles in the first line of host
defense to limit infection after exposure to microorganisms and in
stimulating the clonal response of adaptive immunity (1). Toll-like
receptors (TLRs)1 function as
pattern recognition receptors that recognize conserved motifs on
pathogens (2). To date, 10 members of the TLR family have been
described (3-9). Recent in vitro studies have indicated the
critical roles of TLR2 and TLR4 in recognition of lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid, microbial lipoprotein, mycobacterial lipoarabinomannan, and zymosan from yeast cell walls (10-19). The characteristics of transgenic mice with null allele for
TLR4 provide compelling evidence that TLR4 is a signaling receptor for
LPS (20, 21). The Pro712 It is well recognized that TLRs are critical for cell signaling induced
by broad ranges of microbes and their components. TLRs are thought to
recognize specific molecular patterns of microbial components. One
recent study (26) has revealed that LPS is cross-linked specifically to
TLR4 and MD-2 only when coexpressed with CD14, indicating that LPS
directly binds to each member of the LPS receptor complex. In
vitro and in vivo studies (13, 17, 19, 21, 23)
demonstrate that TLR2 is responsible for cell signaling in response to
S. aureus and its cell wall component, peptidoglycan. PGN
induces the activation of NF- Materials--
iPGN derived from S. aureus was
obtained from Fluka Chemical. LPS from Escherichia coli
O26:B6 and from Salmonella minnesota Re595 were purchased
from Sigma. Recombinant sCD14 expressed in Chinese hamster ovary cells
was prepared as described previously (27). Anti-human TLR2 monoclonal
antibody (mAb 2392) was a kind gift from Genentech (San Francisco, CA).
Anti-human TLR2 monoclonal antibody (mAb TL2.3) was obtained from
Alexis Biochemicals (San Diego, CA).
A Soluble Form of Recombinant Extracellular Toll-like Receptor 2 Domain--
A 2.6-kb cDNA for human TLR2 was obtained by reverse
transcriptase-polymerase chain reaction using RNA isolated from U937 cells. sTLR2 consists of the putative extracellular domain
(Met1-Arg587) and a His6 tag at
the C-terminal end and sTLR2 cDNA was constructed by using PCR. The
sense and antisense primers used were 5'-GGATCCAAAGGAGACCTATAG-3' and
5'-TTAGTGATGGTGATGGTGATGCCTGTGACATTCCGACAC-3', respectively. The sTLR2
cDNA was subcloned into pVL1393 plasmid vector using BamHI and NotI sites. The recombinant plasmid
constructed was confirmed by a combination of restriction enzyme
mapping and DNA sequencing. The sTLR2 protein was expressed in the
baculovirus insect cell expression system using the methods described
by O'Reilly et al. (28). Monolayers of Spodopera
frugiperda (Sf-9) cells were cotransfected with linearized virus
DNA (BaculoGold, Pharmingen) and the pVL1393 plasmid vector containing
cDNA for sTLR2. Viral titers were amplified to ~5-10 × 107 plaque-forming units/ml. The recombinant viruses were
used to infect monolayers of Tni cells in serum-free medium
at a multiplicity of 2. After a 3-day incubation, the sTLR2 protein was
purified from the medium using a column of nickel-nitrilotriacetic acid beads (Qiagen, Valencia, CA) by the method described previously (27).
Protein Analysis--
The protein concentrations were determined
using a bicinchoninic assay (BCA; Pierce). The proteins were analyzed
by SDS-PAGE (12.5% gel) under reducing conditions according to
the method of Laemmli (29) and were stained with Coomassie Brilliant
Blue R-250.
Preparation of Anti-sTLR2 IgG and Anti-CD14 IgG--
Antisera
against sTLR2 and sCD14 were raised in rabbits. sTLR2 (100 µg) or
sCD14 (100 µg) was emulsified in TiterMax® Gold (CytRx Corp.) and
given intradermally. After 3 weeks, a booster with 80 µg of sTLR2 or
sCD14 in the same adjuvant was injected. The blood samples were
collected 2 weeks later, and IgG was isolated from the sera using a
protein G-Sepharose 4FF column (Amersham Biosciences).
Binding of sTLR2 and sCD14 to iPGN and LPS Coated onto Microtiter
Wells--
The binding of sTLR2 and sCD14 to iPGN and LPS was
performed by the method described previously for the binding of sCD14
to LPS (27, 30). The indicated amount of iPGN or LPS in 20 µl of
ethanol was added onto microtiter wells (Immulon 1B, Dynex), and the
solvent was evaporated in ambient air. Nonspecific binding was blocked
with 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2, and 5% (w/v)
bovine serum albumin (buffer A). The indicated concentrations of sTLR2
or sCD14 (50 µl/well) in the buffer A were then added and incubated
at 37 °C for 6 h. After the incubation, the wells were washed
with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100
and were then incubated for 1 h with 30 µg/ml anti-sTLR2 IgG or
anti-sCD14 IgG in the same buffer, followed by incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000) for
1 h. The peroxidase reaction was finally performed using
o-phenylenediamine as a substrate after washing the wells
with PBS containing 0.1% (v/v) Triton X-100. The binding of the
protein to iPGN or LPS was detected by measuring absorbance at 492 nm.
Binding Study of sTLR2 and sCD14 to iPGN by
Sedimentation--
Glass tubes that had been coated with silicone
(SIL-COAT5, IWAKI, Japan) were used in the experiments. The preparation
of 3 µg of sTLR2 or sCD14 in 100 µl of the buffer A was first
centrifuged at 2,000 × g for 5 min. The supernatant
was collected and mixed with 30 µg of iPGN in a fresh silicone-coated
tube. The mixture of protein and iPGN was incubated at 37 °C for
3 h with gentle shaking. After the incubation, the mixture was
centrifuged at 2,000 × g for 5 min, and the
supernatant was removed. The pellet was suspended with 300 µl of PBS
containing 0.1% (v/v) Triton X-100 and centrifuged again at 2,000 × g for 5 min, and the supernatant was removed. The washing
step was repeated three times. The pellet obtained by the final
centrifugation was suspended in 30 µl of the sampling buffer for
SDS-PAGE and boiled for 5 min. sTLR2 or sCD14 that had cosedimented
with iPGN was detected by immunoblotting analysis.
Immunoblot--
The sample was electrophoresed and transferred
to polyvinylidene difluoride membranes. Nonspecific binding was blocked
by incubating the membrane with 20 mM Tris buffer (pH 7.4)
containing 0.15 M NaCl, 3% (w/v) skim milk, and 0.05%
(v/v) Tween 20 (buffer B) for 30 min. The membranes were then incubated
with anti-sTLR2 or anti-sCD14 IgG (10 µg/ml) in the buffer B for
3 h at room temperature. After washing the membranes with the
buffer B, they were further incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG. The proteins that
cosedimented with iPGN were finally visualized using SuperSignal®
West Pico Chemiluminescent Substrate (Pierce) according to the
manufacturer's instructions.
NF- Binding of 125I-Labeled sTLR2 to iPGN--
sTLR2 was
iodinated by the method of Bolton and Hunter (31) using Bolton-Hunter
reagent (Amersham Biosciences). The specific radioactivity ranged from
278 to 293 cpm/ng, and more than 85% of the radioactivity was
precipitated by treatment with 10% (w/v) trichloroacetic acid. The
binding study with 125I-sTLR2 was performed by
sedimentation assay. The indicated concentrations of
125I-sTLR2 were incubated with 1 µg of iPGN (100 µl/tube) in the buffer A at 37 °C for 3 h. After the
incubation, the reaction mixture was transferred into the polypropylene
tube and was centrifuged at 10,000 × g for 5 min. The
supernatant was removed, and the resultant pellet was washed three
times with 500 µl of PBS containing 0.1% (v/v) Triton X-100. The
final pellet was resuspended with PBS containing 0.1% (v/v) Triton
X-100, and the suspension was transferred into a new tube. The
radioactivity of 125I-sTLR2 bound to the iPGN pellet was
measured using a Binding of sCD14 to sTLR2--
Fifty microliters of sTLR2 (10 µg/ml) or bovine serum albumin (10 µg/ml) was added onto microtiter
wells and incubated at 4 °C overnight. After blocking the wells with
the buffer A, the indicated concentrations of sCD14 (50 µl/well) in
the buffer A were added and incubated at 37 °C for 3 h. After
the incubation, the wells were washed with PBS containing 3% (w/v)
skim milk and 0.1% (v/v) Triton X-100, and the binding of sCD14 was
detected using anti-sCD14 IgG.
Effect of sTLR2 on iPGN-induced IL-8 Secretion from U937
Cells--
U937 cells (5 × 105 cells) were
differentiated by incubation with 10 nM phorbol
12-myristate 13-acetate in RPMI 1640 medium containing 10% fetal calf
serum for 24 h. The cells were further incubated in the absence of
phorbol 12-myristate 13-acetate in RPMI 1640 medium containing 10%
fetal calf serum for 24 h. The cells were stimulated in serum-free
RPMI medium for 6 h with 1 µg/ml iPGN that had been preincubated
for 30 min with sTLR2 (0-1 µg/ml). After the incubation,
concentrations of IL-8 secreted from U937 cells were determined by
enzyme-linked immunosorbent assay using an OptEIATM Human
IL-8 Set (Pharmingen, San Diego, CA) according to the
manufacturer's instructions.
Recombinant Proteins--
Approximately 190 µg of sTLR2 was
obtained from 450 ml of culture medium. The N-terminal sequence of
sTLR2 determined by an Applied Biosystems Procise 492 sequencer was
ESSNQASLSXDRNGIXKGSS. This result was
coincident with the deduced amino acid sequence of TLR2 starting at
Glu21. Electrophoretic analysis revealed that sTLR2
exhibited a single band with a molecular mass of ~75 kDa (Fig.
1A). sCD14 migrated as broad
bands with 46-56 kDa, as described previously (27, 32).
sTLR2 Directly Binds to iPGN--
sCD14 has been shown to bind
soluble PGN as well as LPS (33). We investigated the binding of sTLR2
and sCD14 to iPGN coated onto microtiter wells. When 0-5 µg/ml of
sTLR2 and sCD14 were incubated with solid phase iPGN (20 µg/well),
both proteins bound to iPGN in a concentration-dependent
manner (Fig. 2A).
Concentration-dependent binding of sTLR2 to iPGN increased with
the increasing amounts of iPGN (Fig. 2B). The binding of
sTLR2 to O26:B6 LPS or Re LPS was also examined compared with iPGN
binding (Fig. 2C). sTLR2 avidly bound to 20 µg/well iPGN,
but the binding of sTLR2 to 20 µg/well LPS was clearly weak. The
amount of sTLR2 binding to O26:B6 or Re LPS was less than 13% of that
to iPGN.
Another binding study was also performed by sedimentation, because we
used insoluble PGN. To examine whether iPGN was sedimentable by
centrifugation at 2,000 × g for 5 min, we determined
the abilities of the supernatant and the pellet obtained by the
centrifugation to induce tumor necrosis factor-
After the mixture of sTLR2 or sCD14 and iPGN suspension was incubated
and centrifuged, the proteins that had cosedimented with iPGN were
analyzed by immunoblotting using anti-sTLR2 or anti-sCD14 IgG (Fig.
1B). iPGN cosedimented sTLR2 or sCD14. When sTLR2 or sCD14
was incubated without iPGN, none of them was sedimentable. When
surfactant protein A, which does not bind to iPGN (34), was used as a
negative control, iPGN failed to cosediment this protein.
The binding study with 125I-labeled sTLR2 was
also performed by sedimentation assay (Fig.
3). sTLR2 exhibited
concentration-dependent and saturable binding. When the
molecular mass of sTLR2 was estimated as 75 kDa (Fig. 1A),
the binding of sTLR2 to iPGN has an apparent Kd of
67 × 10 Effect of sTLR2 on iPGN-induced NF- Anti-TLR2 mAb 2392 but Not mAb TL2.3 Inhibits the Binding of sTLR2
to iPGN--
Anti-TLR2 monoclonal antibody (mAb 2392), whose epitope
is localized at the extracellular TLR2 domain (35), has been shown to
inhibit secretion of tumor necrosis factor- sCD14 Interacts with sTLR2 and Facilitates the Binding of sTLR2 to
iPGN Coated onto Microtiter Wells--
We next investigated the
interaction of sCD14 with sTLR2. When 0-20 µg/ml of sCD14 were
incubated with sTLR2 coated onto microtiter wells, sCD14 bound to sTLR2
in a concentration-dependent manner (Fig.
6A). When sTLR2 and sCD14 were
preincubated together, the binding of sTLR2 to iPGN increased by 23%
compared with that of sTLR2 alone (Fig. 6B). Coincubation of
these proteins also enhanced the binding of sCD14 to iPGN by 27% (Fig.
6C). These results indicate that sCD14 can interact with
sTLR2 and that coincubation of sTLR2 and sCD14 enhances the binding of
each protein to iPGN.
In this report we demonstrated that the extracellular domain of
TLR2 directly binds PGN. To determine the direct interaction of TLR2
with PGN, we generated the soluble form of recombinant TLR2 possessing
only the putative extracellular domain. sTLR2 bound to iPGN coated onto
microtiter wells in a concentration-dependent manner.
Because we were unable to quantify the amount of iPGN binding to the
wells, we conducted the binding assay in solution with sedimentable
iPGN. Consistent with the microtiter well binding, sTLR2 cosedimented
with iPGN. Taken together, these results clearly demonstrate that the
extracellular domain of TLR2 directly binds to iPGN.
When the binding of sTLR2 to iPGN was compared with that to LPS (Fig.
2C), sTLR2 exhibited a measurable but very weak binding to
LPS. The small amount of sTLR2 binding to LPS may have been due to the
contaminants contained in the LPS preparation, because one recent study
(13) indicates that commercial preparation of LPS contained low levels
of highly bioactive contaminants, endotoxin protein, which is
responsible for the TLR2-mediated signaling observed upon LPS
stimulation, and that repurification of LPS eliminates cell signaling
mediated through TLR2. The current study indicating the avid binding of
sTLR2 to iPGN is essentially consistent with the previous in
vivo and in vitro studies (17, 21) showing that TLR2
functions as a physiological receptor for PGN but not for LPS.
Coincubation of sTLR2 with iPGN partially neutralized both iPGN-induced
NF- Anti-TLR2 mAb 2392 that had been generated against the extracellular
TLR2 domain (35) was reported to inhibit PGN-induced cytokine release
from THP1-CD14 cells (36). Likewise, this study showed that mAb 2392 inhibited NF- One recent study (26) has revealed that LPS is cross-linked
specifically to TLR4 and MD-2 only in the cells expressing CD14, indicating the direct binding of LPS to TLR4. In addition, the study
has shown that LPS is cross-linked to TLR2 even when CD14 and MD-2 are
not transfected. This study indicates that TLR2 directly binds to PGN
in the absence of CD14. These studies suggest that TLR2, unlike TLR4,
does not require CD14 to bind the ligand, supporting the idea that the
mechanism of TLR2-ligand interaction is different from that of TLR4. In
conclusion, we demonstrated that the extracellular TLR2 domain directly
binds PGN. The conclusion obtained from this study using the cell-free
system is consistent with that obtained from the study using the cells
with transient transfection, showing that TLR directly binds its ligand
and that the TLR-ligand interaction does not require cellular
energy (26).
B activation in TLR2-transfected HEK 293 cells and the
iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2
monoclonal antibodies, which blocked iPGN-induced NF-B activation in
TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In
addition, we found that sCD14 interacted with sTLR2 and increased the
binding of sTLR2 to iPGN. From these results, we conclude that the
extracellular TLR2 domain directly binds to PGN.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His mutation in the
Tlr4 gene causes a defective response to LPS in C3H/HeJ mice
(22). TLR2 (
/) mice were highly susceptible to Staphylococcus
aureus infection (23). Macrophages from TLR2-deficient mice
responded to LPS but were unable to produce proinflammatory cytokines
in response to PGN (21), indicating that PGN-induced cell activation is
mediated by TLR2. In addition, TLR5 and TLR9 have recently been shown
to be involved in recognition of bacterial flagellin and DNA,
respectively (24, 25).
B in HEK 293 cells expressing TLR2 in
the absence of CD14, although coexpression of CD14 enhances TLR2-mediated signaling (17). This suggests that TLR2 can interact with
PGN regardless of whether CD14 is expressed. The specific objective of
this study was to determine whether TLR2 directly binds PGN. In this
study we generated a soluble form of recombinant TLR2 (sTLR2) lacking
the putative intracellular and transmembrane domains and conducted a
binding study using sTLR2 and insoluble PGN (iPGN) derived from
S. aureus. This study clearly demonstrated that the
extracellular TLR2 domain binds iPGN.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Reporter Assay--
A 2.6-kb TLR2 cDNA was subcloned
into pcDNA3.1(+) (Invitrogen). Activation of NF-B was measured
as described previously (14, 17). HEK 293 cells were plated at 1 or
1.5 × 105 cells/well in 24-well plates on the day
before transfection. The cells were transiently transfected by
FuGENETM 6 transfection reagent (Roche Molecular
Biochemicals) with 30 or 100 ng of an NF-B reporter construct
(pNF-B-Luc; Stratagene) and 3.5 or 10 ng of a construct that
directed expression of Renilla luciferase under the control
of the constitutively active thymidine kinase promoter (pRL-TK;
Promega), together with 150 or 200 ng of transfectant gene. Forty-eight
hours after transfection, the cells were stimulated with 0.1, 1.0, or
10 µg/ml iPGN in serum-free medium for 6 h, and luciferase
activity was measured by using the dual luciferase reporter assay
system (Promega) according to the manufacturer's instructions. In some
experiments the transfected cells were preincubated with monoclonal
antibody or control mouse IgG at 37 °C for 30 min. To examine the
effect of sTLR2 on iPGN-induced NF-B activation, iPGN was
preincubated with sTLR2 (0-10 µg/ml) for 30 min before incubation
with the cells.
-radiation counter. Specific binding was determined
by subtracting the binding obtained in the absence of iPGN from that in
the presence of iPGN.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electrophoretic analysis of sTLR2 and its
binding to iPGN. A, electrophoretic analysis of sTLR2
and sCD14. sTLR2 (3.3 µg) expressed in insect cells and sCD14 (3.0 µg) expressed in Chinese hamster ovary cells were subjected to
SDS-polyacrylamide gel electrophoresis under reducing conditions. The
proteins were visualized by Coomassie Brilliant Blue staining.
B, binding analysis of sTLR2 to iPGN by sedimentation. 3 µg of sTLR2 or sCD14 was incubated with or without 30 µg of iPGN
(100 µl/tube) at 37 °C for 3 h. The mixture was centrifuged,
the pellet obtained was electrophoresed, and the protein cosedimenting
with iPGN was detected by immunoblot using anti-sTLR2 or anti-sCD14 IgG
as described under "Experimental Procedures."
st., standard; cont., control; mol.
mass, molecular mass.
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Fig. 2.
sTLR2 binds to iPGN coated onto microtiter
wells. A, concentration-dependent binding of
sTLR2 and sCD14 to iPGN. The indicated concentrations of sTLR2
(closed circles) and sCD14 (open circles) were
incubated with iPGN (20 µg/well) coated onto microtiter wells at
37 °C for 6 h. The binding of sTLR2 or sCD14 to iPGN was
detected using anti-sTLR2 or anti-sCD14 IgG as described under
"Experimental Procedures." B, binding of sTLR2 to
various amounts of iPGN. The indicated concentrations of sTLR2 were
incubated with 0 (closed squares), 2 (open
triangles), 5 (closed triangles), 10 (open
circles), or 20 µg (closed circles) of iPGN coated
onto microtiter wells. The binding of sTLR2 to iPGN was detected using
anti-sTLR2 IgG as described under "Experimental Procedures."
C, binding of sTLR2 to iPGN and LPS. The indicated
concentrations of sTLR2 was incubated with iPGN (closed
triangles), O26:B6 LPS (closed circles), or Re LPS
(open circles) coated onto microtiter wells at 37 °C for
6 h. The binding of sTLR2 to iPGN or LPS was detected using
anti-sTLR2 IgG as described under "Experimental Procedures."
secretion from U937
cells. Tumor necrosis factor- secretion was measured using the L929
bioassay as described previously (30). The pellet fraction contained 99.3% (mean of three experiments) of tumor necrosis
factor--inducing activity, indicating that almost all of the
iPGN was sedimentable by the centrifugation.
9 M. A maximum of ~40 ng (0.53 pmol) of sTLR2 was assumed to bind to 1 µg of iPGN. Taken together,
these data clearly demonstrate that sTLR2 directly binds to iPGN.
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Fig. 3.
Binding of 125I-labeled sTLR2 to
iPGN. The indicated concentrations of 125I-sTLR2 (100 µl/tube) were incubated with 1 µg of iPGN at 37 °C for 3 h.
sTLR2 bound to iPGN was separated from unbound protein by
centrifugation at 10,000 × g. After the iPGN pellet
was washed, the radioactivity of 125I-sTLR2 bound to the
iPGN pellet was determined as described under "Experimental
Procedures." The inset shows the Scatchard plot. The data
shown are the means of two experiments.
B Activation and IL-8
Secretion--
We examined the effect of sTLR2 on iPGN-induced NF-B
activation in TLR2-transfected HEK 293 cells. Coincubation of iPGN (100 and 1000 ng/ml) and sTLR2 (0.1-10 µg/ml) attenuated the activities of NF-B (Fig. 4A). sTLR2 at
1 and 10 µg/ml reduced the NF-B activity by ~40-50%. We also
examined the effect of sTLR2 on iPGN-induced IL-8 secretion in U937
cells. When differentiated U937 cells were stimulated with iPGN (1 µg/ml) that had been preincubated with sTLR2 (0-1 µg/ml), the IL-8
secretion was decreased in a manner dependent upon the sTLR2
concentrations (Fig. 4B). sTLR2 at 1 µg/ml reduced the
iPGN-induced IL-8 secretion by 36%.
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Fig. 4.
sTLR2 partially attenuates iPGN-induced
NF- B activation and IL-8 secretion.
A, effect of sTLR2 on iPGN-induced NF-B activity in wild
type TLR2-transfected HEK 293 cells. Luciferase activities were
determined in 1 × 105 HEK 293 cells plated in 24-well
plates and transfected with the expression plasmid vector containing
wild type TLR2 cDNA (150 ng) together with an NF-B reporter
plasmid (pNF-B-Luc, 30 ng) and Renilla luciferase control
reporter plasmid (pRL-TK, 3.5 ng). Forty-eight hours after
transfection, the cells were stimulated for 6 h with iPGN (100 or
1000 ng/ml) that had been preincubated with sTLR2 (0-10 µg/ml) at
37 °C for 30 min before harvest. The data shown are the means ± S.E. of three experiments. B, effect of sTLR2 on
iPGN-induced IL-8 secretion in U937 cells. Differentiated U937 cells
(5 × 105 cells) were stimulated for 6 h with 1 µg/ml iPGN that had been preincubated with sTLR2 (0-1 µg/ml) at
37 °C for 30 min. After the incubation, the concentrations of IL-8
secreted from U937 cells were determined by enzyme-linked immunosorbent
assay.
and IL-8 elicited by PGN
in macrophage cell line THP-CD14, a monocytic cell line that
constitutively expresses CD14 (36). Another mAb TL2.3 also recognizes
the extracellular TLR2 domain and has been shown to immunostain
peripheral blood mononuclear cells (37). When iPGN was incubated with
TLR2-transfected HEK 293 cells in the presence of mAb 2392, the
antibody inhibited the iPGN-induced NF-B activity in a
concentration-dependent manner (Fig.
5A). Coincubation of 10 µg/ml mAb 2392 with TLR2-transfected cells in the presence of 10 µg/ml iPGN significantly reduced the NF-B activity to the level of
~15% of that in the absence of antibody. This is essentially consistent with the results observed for cytokine secretion in THP-CD14
cells (36) and supports the idea that the epitope of mAb 2392 is
located at the TLR2 region that is involved in PGN recognition.
However, 10 µg/ml mAb TL2.3 failed to attenuate the iPGN-induced
NF-B activation. The effects of mAbs on the binding of sTLR2 to iPGN
were also examined (Fig. 5B). mAb 2392 significantly decreased the binding of sTLR2 to iPGN in a dose-dependent
manner. The binding of sTLR2 to iPGN in the presence of 10 µg/ml mAb
2392 was reduced to the level of 35% of that in the absence of the mAb. In contrast, 10 µg/ml mAb TL2.3 did not attenuate the binding of
sTLR2 to iPGN. Its level was almost comparable with that obtained in
the presence of control mouse IgG. The results support the conclusion
obtained from the direct binding assay that the extracellular TLR2
domain directly binds iPGN.
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Fig. 5.
Anti-TLR2 monoclonal antibody 2392 but not
TL2.3 inhibits the binding of sTLR2 to iPGN. A, effects of
anti-TLR2 mAbs 2392 and TL2.3 on iPGN-induced NF- B activation.
Luciferase activities were determined in 1.5 × 105
HEK 293 cells plated in 24-well plates and transfected with the
expression plasmid vector containing wild type TLR2 cDNA (200 ng)
together with an NF-B reporter plasmid (pNF-B-Luc, 100 ng) and
Renilla luciferase control reporter plasmid (pRL-TK, 10 ng).
Forty-eight hours after transfection, the cells were preincubated with
the indicated concentrations of anti-TLR2 mAb 2392, mAb TL2.3, or
control mouse IgG (mIgG) at 37 °C for 30 min and were further
stimulated with 10 µg/ml iPGN for 6 h. The results are expressed
as percentages of luciferase activity obtained from iPGN stimulation in
the absence of antibody. The data shown are mean ± S.E. of three
experiments. *, p < 0.005, compared with iPGN
stimulation without antibody. B, effects of anti-TLR2 mAbs
2392 and TL2.3 on the binding of sTLR2 to iPGN. sTLR2 (5 µg/ml) was
preincubated with the indicated concentrations of mAb 2392, mAb TL2.3,
or control mouse IgG (mIgG), and the mixture of sTLR2 and antibody was
further incubated with iPGN (20 µg/well) coated onto microtiter wells
at 37 °C for 6 h. The binding of sTLR2 to iPGN was detected by
anti-sTLR2 rabbit IgG as described under "Experimental Procedures."
The results are expressed as percentages of the sTLR2 binding to iPGN
in the absence of antibody. The mean absorbance (492 nm) of the sTLR2
binding to iPGN in the absence of antibody was 0.56. The data shown are
the means ± S.E. of three experiments. **, p < 0.01, compared with iPGN stimulation in the absence of
antibody.
View larger version (11K):
[in a new window]
Fig. 6.
sCD14 interacts with sTLR2 and facilitates
the binding of sTLR2 to iPGN coated onto microtiter wells.
A, binding of sCD14 to sTLR2 coated onto microtiter wells.
The indicated concentrations of sCD14 were incubated with sTLR2 or
bovine serum albumin coated onto microtiter wells at 37 °C for
3 h. The binding of sCD14 was detected using anti-sCD14 IgG as
described under "Experimental Procedures." B and
C, binding of sTLR2 or sCD14 to iPGN. sTLR2 (5 µg/ml) and
sCD14 (5 µg/ml) were preincubated together at 37 °C for 30 min,
and the mixture (50 µl) was further incubated with iPGN (20 µg/well) coated onto microtiter wells at 37 °C for 6 h. The
binding of sTLR2 or sCD14 to solid phase iPGN was detected by
anti-sTLR2 IgG or anti-sCD14 IgG, respectively, as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation in TLR2-transfected HEK 293 cells and IL-8 secretion
in U937 cells (Fig. 4). It is likely that the interaction of sTLR2 with
iPGN causes the decreased binding of iPGN to TLR2 expressed on cell
surfaces, resulting in the attenuated cell signaling. The naturally
occurring soluble form of mouse TLR4 that is expressed by alternatively
spliced mouse TLR4 mRNA has been shown to attenuate but not
completely inhibit LPS-elicited NF-B activities to the level of
50-60% of those observed for controls (38). These results indicate
that the extracellular TLR domain alone does not explain all the
activity of TLR-ligand interaction. Although it is unclear why the
soluble form of TLRs did not completely inhibit cell signaling, it is
possible to assume that the wild type TLRs and the soluble form of the
extracellular TLR domain lacking the transmembrane and cytoplasmic
domains may differ in affinities for the ligands. Actually the affinity
(the apparent Kd = 67 × 109
M) of sTLR2 for iPGN was not so high as was expected. It is
uncertain whether wild type TLR2 on cell surfaces interacts with PGN
with the same affinity as sTLR2 constructed. Partial attenuation of iPGN-induced signaling and cytokine secretion by the soluble form of
TLR may be a consequence of the binding affinity of the soluble form, which is different from membrane-bound form. Other factors including sCD14 and other classes of TLR may be required for the acquisition of high affinity binding. As shown in Fig. 6, sCD14 interacted with sTLR2 and facilitated the binding of sTLR2 to solid
phase iPGN. These results are consistent to the previous studies
indicating that PGN-induced NF-B activation is increased by
cotransfection of membrane bound CD14 with TLR2 in HEK 293 cells (17,
39). Taken together, these results support the idea that the
PGN-induced cellular response is closely related with the direct
binding of PGN to the extracellular TLR2 domain.
B activity induced by iPGN in TLR2-transfected 293 cells (Fig. 5A). mAb 2392 also significantly decreased the
binding of sTLR2 to solid phase iPGN (Fig. 5B). Although the
epitope for this monoclonal antibody has not been identified, the
extracellular TLR2 region contiguous to the antibody epitope may be
critical for ligand recognition. In contrast, another mAb TL2.3 failed
to inhibit iPGN-induced NF-B activation and sTLR2 binding to iPGN
(Fig. 5). These results may give specificity and biological relevance
to the experiments. The study supports the idea that the direct binding
of the extracellular TLR2 domain initiates iPGN-induced cell signaling.
| |
FOOTNOTES |
|---|
* This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and by the Novartis Foundation (Japan).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Sapporo Medical University School of Medicine, South-1 West-17,
Chuo-ku, Sapporo 060-8556, Japan. Tel.: 81-11-611-2111; Fax:
81-11-611-2236; E-mail: kurokiy@sapmed.ac.jp.
Published, JBC Papers in Press, May 1, 2002, DOI 10.1074/jbc.M107057200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
TLR, Toll-like
receptor;
sTLR2, a soluble form of extracellular TLR2 domain;
sCD14, soluble CD14;
PGN, peptidoglycan;
iPGN, insoluble PGN;
LPS, lipopolysaccharide;
NF-B, nuclear factor-B;
mAb, monoclonal
antibody;
HEK, human embryonic kidney;
PBS, phosphate-buffered saline;
IL, interleukin.
| |
REFERENCES |
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RESULTS
DISCUSSION
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