From The Scripps Research Institute, Department of
Immunology and the § Department of Neuropharmacology,
La Jolla, California 92037
Received for publication, October 6, 2000, and in revised form, March 2, 2001
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ABSTRACT |
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The structural features of some proteins of the
innate immune system involved in mediating responses to microbial
pathogens are highly conserved throughout evolution. Examples include
members of the Drosophila Toll (dToll) and the mammalian
Toll-like receptor (TLR) protein families. Activation of
Drosophila Toll is believed to occur via an endogenous
peptide rather than through direct binding of microbial products to the
Toll protein. In mammals there is a growing consensus that
lipopolysaccharide (LPS) initiates its biological activities through a
heteromeric receptor complex containing CD14, TLR4, and at least one
other protein, MD-2. LPS binds directly to CD14 but whether LPS then
binds to TLR4 and/or MD-2 is not known. We have used transient
transfection to express human TLRs, MD-2, or CD14 alone or in different
combinations in HEK 293 cells. Interactions between LPS and these
proteins were studied using a chemically modified, radioiodinated LPS
containing a covalently linked, UV light-activated cross-linking group
(125I-ASD-Re595 LPS). Here we show that LPS is cross-linked
specifically to TLR4 and MD-2 only when co-expressed with CD14. These
data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.
Bacterial endotoxin (lipopolysaccharide,
LPS)1 okonp61 is a complex
glycolipid composed of a hydrophilic polysaccharide moiety and a
hydrophobic domain known as lipid A (1). LPS is an outer membrane
constituent of all Gram-negative bacteria where it has indispensable
barrier functions. LPS is also a potent activator of innate immune
responses that result in the production of pro- and anti-inflammatory
mediators from myeloid lineage and other cell types (2). LPS-induced
cell activation depends on the presence of three proteins comprising a
multiprotein cell surface receptor complex here termed the LPS receptor
complex. One essential protein of the LPS receptor complex is CD14 (3),
a 55-kDa glycoprotein present in soluble form (sCD14) in blood or as a
membrane-bound form (mCD14) in myeloid lineage cells. This latter form
is attached to the outer leaflet of the cell membrane via a
glycosylphosphatidylinositol anchor. Multiple lines of
biochemical and genetic evidence support the contention that CD14
principally acts to bind LPS and does not participate in signaling
directly. Thus, others and we postulated that there must be at least
one transmembrane protein that acts in concert with CD14 (2). This
putative transmembrane protein is now identified as a member of the
mammalian Toll-like receptor (TLR) family and is TLR4 (4, 5). Genetic
and biochemical studies suggest that TLR4 plays an important role in
LPS signaling under physiological conditions. Positional cloning and
sequencing of the lpsd locus localized the
defect to the tlr4 gene (6). The importance of TLR4 in LPS
signaling is further supported by the fact that TLR4-deficient mice are
LPS hyporesponsive but respond normally to products of Gram-positive
organisms (7). MD-2 is another protein that appears to be important in
LPS signaling (8). However, its function in the LPS receptor complex is
currently unknown.
TLR2 is a signaling receptor for a variety of microbial products that
in some cases require CD14 for maximum activation (9). Although TLR2
was initially thought to function as an LPS receptor current evidence
suggests that it does not play a major role in the physiological
response to LPS (9-11). Nonetheless a recent publication provides data
showing that when TLR2 is overexpressed in 293 cells it mediates cell
activation induced by a variety of purified LPS isolates (12). It thus
appears that TLR2 is more promiscuous with respect to ligand
recognition and that LPS must be added to the list of TLR2 ligands.
Numerous reports describe the direct binding of LPS to CD14 (3,
13-15). The question of whether LPS binds to, or even is in close
proximity to the other proteins of the LPS receptor remains unanswered.
Here we have used radioiodinated Re595 LPS with a covalently attached
UV-activated phenylazide that is capable of cross-linking to nearby
proteins in order to characterize interactions between LPS and the LPS
receptor complex at the cell surface. Our results show that LPS is
brought into close proximity to TLR4 only when it is present as an
LPS·CD14 complex and when CD14 and TLR4 are co-expressed with
MD-2.
Cell Culture and Transfection--
Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 units/ml penicillin, and 10 µg/ml streptomycin. 3.5 × 106 cells in 10-cm Petri dishes were transfected using
LipofectAMINE Reagent Plus (Life Technologies, Inc., Gaithersburg, MD).
The total amount of DNA was kept constant by supplementing pFLAG-CMV1 vector DNA (Eastman Kodak Co., Rochester, NY).
Reagents--
Murine anti-human TLR4 monoclonal antibodies were
from Dr. K. Miyake. The following reagents were prepared as described
in the references: Re595 LPS (16), rabbit LBP (17), and recombinant human soluble CD14 (18). Mouse monoclonal antibodies against CD14
(28C5, 63D3, and 18E12) were a gift from A. Moriarty and D. Leturcq
(R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA).
M2 anti-FLAG monoclonal antibody was from Sigma. Escherichia coli LCD25 LPS and 0111:B4 LPS were from List Biological Labs (Campbell, CA). E. coli 055:B5 LPS was from Sigma. Protein
A-Sepharose was from Amersham Pharmacia Biotech (Piscataway, NJ). In
order to mitigate concerns about protein contaminants the Re595 LPS was
also repurified according to the methods of Hirschfeld et al. (10). This material was also used to prepare
125I-ASD-Re595 LPS; results obtained with the
125I-ASD derivative of the repurified Re595 LPS were not
distinguishable from the parent LPS.
Mammalian Expression Constructs--
Human CD14 cDNA was
cloned in pRc/RSV vector as described (18). The cDNAs for human
TLR2 and TLR4 were gifts from Drs. P. Godowski and C. Janeway,
respectively. The cDNAs for Drosophila Toll and human
TLR1 were gifts from Dr. Alan Aderem. FLAG-dToll was cloned in
pFLAG-CMV1 using HindIII/BamHI restriction sites. Expression vectors for FLAG-MD-2 was engineered by introducing a
NotI/SmaI DNA fragment into pFLAG-CMV1 plasmid,
in which the prepro trypsin leader sequence precedes an
NH2-terminal FLAG epitope.
125I-ASD-Re595 LPS Cross-linking Assays--
293
cells cultured in 10-cm Petri dishes were transfected and harvested 2 days after transfection. Cells were washed 2 times with 50 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM
EDTA, pH 7.4, and 2 times with DMEM containing 50 mM HEPES,
pH 7.4. Cells were pelleted and resuspended in Dulbecco's modified
Eagle's medium containing 50 mM HEPES, pH 7.4, and 2.5%
endotoxin-free human bovine serum albumin (Serologicals Proteins Inc.,
Kankakee, IL). LBP·LPS complexes were formed and utilized as
previously described (19). Briefly, 25 µg/ml rLBP and 2.5 µg/ml
125I-ASD-Re595 LPS were incubated in a solution containing
25 mM HEPES, pH 7.4, and 2.5 mM EDTA, pH 8, 10 min at 37 °C. LBP·LPS complexes were added to cells, incubated 10 min at 37 °C, and photolyzed with a 253-nm UV light for 4 min on
ice. A typical incubation reaction contained a final concentration of
250 ng/ml 125I-ASD Re595 LPS and 2.5 µg/ml rLBP. When
metabolic inhibition was specified, cells were incubated for 30 min at
37 °C in binding buffer containing 10 mM sodium azide, 2 mM sodium fluoride, and 5 mM deoxyglucose prior
to addition of LBP·LPS complexes.
Preparation of Cell Lysates--
Cells were washed extensively
and lysed in 1 ml of lysis buffer containing 50 mM Hepes,
100 mM NaCl, 2 mM EDTA, 10% glycerol, 1%
Nonidet P-40, 14 µM pepstatin A, 100 µM
leupeptin, 3 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 100 units/ml aprotinin, 100 mM sodium fluoride. After incubation for 30 min on ice,
cell lysates were centrifuged (14,000 rpm, 10 min, 4 °C) and the
supernatants were recovered.
Immunoprecipitation and Western Blot Analysis--
Cell lysates
were pre-cleared 3 times for 20 min at 4 °C with 60 µl of protein
A-Sepharose beads, and mixed with 30 µg of M2 monoclonal antibodies
for 3 h at 4 °C under constant agitation. Immune complexes were
allowed to bind to 60 µl of protein A-Sepharose beads overnight,
beads were washed 3 times with lysis buffer and the washed beads
resuspended in 100 µl of Laemmli buffer and boiled for 10 min.
Aliquots from 20 µl of the original mixtures were separated on 12%
SDS-PAGE and transferred to nitrocellulose membranes. Filters were
blocked with 5% nonfat milk in blocking buffer (TBS-T, 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween
20), and incubated with anti-FLAG antibody for 2 h and with
peroxidase-conjugated secondary antibody for 1 h at ambient
temperature. Specific bands were revealed using the ECL Plus system
(Amersham Pharmacia Biotech). Aliquots of 80 µl of immunoprecipitates
were separated on 12% SDS-PAGE. The gels were dried and the
radioactivity associated with protein was quantitated with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Each experiment
described here was performed at least three times. Here we show
individual representative experimental data.
Flow Cytometry Analysis--
293 cells were plated at a density
of 5 × 105 cells/well in 6-well plates, and
transfected with the indicated plasmids together with pEGFP-N3 vector
(CLONTECH Laboratories, Inc., Palo Alto, CA) for 2 days. Subsequently, cells were harvested, washed twice in
phosphate-buffered saline containing 1% fetal calf serum and 0.1%
NaN3 and incubated with anti-FLAG or HTA125 (10 µg/ml
each) for 45 min at 4 °C. After 2 washes, cells were labeled for 45 min with phycoerythrin-conjugated goat anti-mouse antibody (BD PharMingen, San Diego, CA). Cells were then washed twice and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).
LPS Is Cross-linked to TLR4 and MD-2 in a
CD14-dependent Manner--
HEK 293 cells were transfected
with various combinations of DNA encoding wild-type human CD14 and
epitope-tagged (FLAG) versions of TLR4 and MD-2. Two days after
transfection the cells were washed and exposed to a preformed complex
of LBP and 125I-ASD-Re595 LPS as described (19). After
photolysis, cells were lysed and the lysates treated with anti-FLAG
monoclonal antibody. The resultant immunoprecipitates were subjected to
SDS-PAGE first to reveal radioactive bands by phosphorimaging
(top panels) and then to determine TLR4 and MD-2 protein
expression (bottom panels) by immunoblotting with anti-FLAG
antibody. Evidence for LPS cross-linking to TLR4 and MD-2 was obtained
by the radiolabeled protein bands noted only when CD14 was expressed
(Fig. 1A, lane 7). We failed to observe binding of LPS to TLR4 or MD-2 when expressed alone (lanes 2 and 4, respectively) or in combination
with CD14 (lanes 3 and 5). When TLR4 and MD-2
were expressed together a barely detectable band corresponding to MD-2
was found (lane 6). In dose-response studies, not shown,
identical labeling patterns were observed even when we reduced the
input LPS concentration to as low as 10 ng/ml. Unless otherwise noted
all experiments were done with 250 ng/ml 125I-ASD-Re595
LPS. Western blotting revealed similar levels of expressed proteins.
MD-2 appears as two species characterized by faster and slower
migrating forms due to differential glycosylation
patterns.2 Although CD14 does
not contain an epitope tag we were unable to fully eliminate it from
anti-FLAG immunoprecipitates despite a pre-clear step and extensive
washing. Several lines of evidence suggest this reflects a minor amount
of nonspecific carryover in the immunoprecipitates. First, quantitation
of the amount of bound LPS showed that LPS bound to TLR4 and MD-2
represented less than 0.1% of total LPS bound to the
CD14·TLR4·MD-2 complex. The remainder is bound to CD14 or other
proteins. This is not surprising given the major function of CD14 to
bind and internalize LPS in a high capacity manner. Second, analysis of
whole cell lysates only revealed the presence of LPS bound to CD14
whereas immunoprecipitation was required to reveal LPS bound to TLR4
and MD-2 (data not shown).
Both mCD14 and sCD14 function to enhance cellular responses to LPS (3,
20-22). We next examined whether sCD14 could substitute for mCD14 in
facilitating LPS binding to TLR4 and MD-2. 293 cells were transfected
with TLR4 and MD-2 either alone or in combination. The cells were then
harvested, extensively washed, and exposed to the preformed LBP·LPS
complex plus sCD14; sCD14 mediated LPS binding to TLR4 and MD-2 only
when both proteins were co-expressed (Fig. 1B, lane 4). No
labeling of TLR4 or MD-2 was noted when these proteins are expressed
alone (lanes 2 and 3). These data suggest that
LPS was delivered from an LBP·LPS complex to a TLR4·MD-2 complex in
a CD14-dependent manner.
To determine whether any other member of the TLR family would bind LPS
in a CD14 and MD-2 dependent fashion, experiments were carried out with
cells expressing TLR4, TLR1, or dToll. 293 cells were transfected with
MD-2 and CD14 either in the presence of TLR4, TLR1, or dToll. Fig.
2A shows a specific binding of
LPS only to TLR4. No binding to TLR1 or dToll could be observed under identical experimental conditions. Expression of each of the proteins was evaluated by immunoblotting (Fig. 2A, bottom panel) and
flow cytometry (Fig. 2B). TLR4 and MD-2 were clearly
detectable at cell surface of 293 cells when expressed alone using
HTA125, a monoclonal anti-TLR4 antibody, or with anti-FLAG antibody.
The co-expression of TLR4 with MD-2 dramatically enhanced the
fluorescence intensity of TLR4. This effect was observed with either
anti-FLAG or HTA125 antibody. Moreover it was dependent on MD-2, since
the introduction of CD14 in the absence of MD-2 had no effect on the fluorescence staining of TLR4 (data not shown). Both TLR1 and dToll
were expressed at cell surface as shown in Fig. 2B
(bottom panels). Unlike our observations with TLR4,
co-expression with MD-2 did not modify cell surface expression of TLR1
or dToll (data not shown).
TLR2 had been reported to be a receptor for Gram-negative LPS (23, 24).
However, these observations resulted in part from trace
contaminants present in the LPS isolates (10, 25). On the other hand
recent work of Dziarski et al. (12) strongly suggests that
TLR2 can recognize some forms of purified, protein-free LPS including
that of Re595 LPS. As part as our exploration of the specificity of the
labeling reaction we asked whether Re595 LPS might also be brought into
close proximity with TLR2 and if so whether CD14 and MD-2 are involved.
We observed cross-linking of Re595 LPS to TLR2 in the absence of CD14
and MD-2 (Fig. 3, lane 2). We
noted that co-expression of CD14 somewhat enhanced the amount of LPS
bound to TLR2 (lane 3). In contrast to our findings with
TLR4, co-expression of MD-2 had no detectable influence on Re595 LPS
cross-linking to TLR2 whether or not CD14 was present (lanes
4 and 5). Moreover, we failed to observe any detectable binding of Re595 LPS to MD-2 regardless of whether TLR2 and/or CD14
were expressed. TLR2 protein expression level was constant in each of
the conditions considered in this experiment (Fig. 3A, bottom
panel) and by flow cytometry analysis (Fig. 3C).
Interestingly, and in contrast to findings with TLR4, expression of
MD-2 failed to enhance cell surface expression of TLR2 (not shown).
Others recently showed that co-expression of TLR2 and MD-2 resulted in increased total expression of each protein in cell lysates when compared with the levels detected with each expressed individually (12). However, the latter study did not determine the surface expression levels so we cannot directly compare our findings.
In order to be certain of the purity of the LPS used in our studies we
re-extracted Re595 LPS according to the methods described in Hirschfeld
et al. (10). We used the repurified LPS to prepare the
radioiodinated derivative for cross-linking studies. The derivatized, repurified Re595 LPS behaved identically in all experiments including the findings of cross-linking to TLR2 as noted above (data not shown).
Thus it is virtually certain that the results we describe here with the
derivatized Re595 LPS solely reflect interactions of LPS and not
protein contaminants with cell surface proteins.
Binding of LPS to CD14 has been shown to be independent of cellular
energy metabolism (26). We next investigated cellular energy
requirements for LPS binding to MD-2 and TLR4. To do this we compared
CD14-dependent binding of Re595 LPS to TLR4 and MD-2 expressed in 293 cells; LPS was added to the cells in the presence and
absence of metabolic inhibitors (Fig. 4).
Addition of sodium azide, sodium fluoride, and 2-deoxyglucose failed to
prevent Re595 LPS cross-linking to TLR4·MD-2. Thus transfer of LPS
from CD14 to the TLR4·MD-2 complex does not require cellular energy.
Similar results were observed with TLR2.
Specificity of Re595 LPS Binding to TLR4 and MD-2--
The lipid A
moiety has been identified as the LPS component responsible for
LPS-induced biological effects through direct interaction with CD14
(1). To determine whether LPS binding to TLR4 and MD-2 is a lipid
A-dependent phenomenon, competition experiments were
carried out in 293 cells transfected with CD14, TLR4, and MD-2. Cells
were preincubated with an excess of various LPS isolates, a partial
lipid A structure known as lipid IVa (27) and lipoteichoic acid for 3 min prior to the addition of 125I-ASD-Re595 LPS (Fig.
5). All LPS from E. coli
serotypes tested, 0111:B4, 055:B5, and LCD25, inhibited binding of
Re595 LPS. The different LPS isolates as well as lipid IVa blocked LPS
binding to TLR4 and MD-2 (Fig. 5, B and C). In
contrast, lipoteichoic acid did not alter Re595 LPS binding to TLR4 and
MD-2. Others have suggested that lipoteichoic acid is a ligand for
TLR2 (28-30). These data support the contention that the interactions
between Re595 LPS and CD14·TLR4·MD-2 are lipid
A-dependent.
To further confirm the importance of CD14 in transfer of LPS to the
TLR4·MD-2 complex, we evaluated 125I-ASD-Re595 LPS
binding to TLR4 and MD-2 in the absence or presence of various
anti-CD14 monoclonal antibodies (Fig. 6).
Three monoclonal antibodies were used; 28C5 known to prevent LPS
binding to both mCD14 and sCD14 (31), 18E12 an antibody which prevents
LPS-induced cell activation but does not block LPS binding to CD14
(26); and 63D3 an antibody that has minimal effects of cell activation and on LPS binding to CD14 (32). Pretreatment with 28C5 markedly inhibited LPS binding to TLR4 and MD-2. Treatment with 18E12 did not
prevent LPS binding to TLR4 and slightly enhanced binding to MD-2. The
antibody 63D3 slightly decreased cross-linking to TLR4 and MD-2. In
totality these results suggest that binding of LPS to CD14 is essential
for the subsequent step(s) that facilitate interactions of LPS with
TLR4 and MD-2. Interestingly 18E12, an anti-human CD14 monoclonal
antibody that prevents cell activation without blocking LPS binding to
CD14 did not prevent LPS interactions with other members of the
receptor complex. This suggests that additional steps subsequent to LPS
binding to TLR4 and MD-2 are required for cell activation. Further
support for this concept derives from studies with anti-TLR4 monoclonal
antibodies. Of the four monoclonal antibodies available to us, HTA405,
HTA414, and HTA1216 all strongly inhibited LPS-induced TNF and IL-8
release from blood cells (25). One, HTA125, failed to show the
inhibitory activity in the same assay system (data not shown). Thus we
used these antibodies to see whether they could modulate LPS binding to
TLR4 and MD-2. Surprisingly none of the inhibitory antibodies reduced
radiolabeling of TLR4 or MD-2. One of the three blocking antibodies,
HTA405, reduced the radiolabeling by about 30% (data not shown)
suggesting it may react at or near an LPS-binding site of TLR4.
Here we have used a radioiodinated derivative of Re595 LPS
substituted with a UV-activated phenylazide to determine whether, after
binding to CD14, we could detect cross-linking of Re595 LPS to other
proteins in the putative LPS receptor complex. Using this approach we
showed that after binding to CD14, Re595 LPS is in close enough
proximity to both TLR4 and MD-2 that cross-linking occurred. These
interactions do not require cellular energy, but are strikingly
facilitated by CD14, as in cellular activation. Anti-CD14 monoclonal
antibodies that block LPS binding to CD14 prevent the subsequent
interactions with TLR4 and MD-2. Interestingly anti-TLR4 monoclonal
antibodies that inhibit LPS-induced cell activation do not prevent LPS
binding to CD14, TLR4, or MD-2. Altogether these data suggest that LPS
binds directly to proteins in the LPS membrane receptor complex but
that binding to TLR4 and/or MD-2 is not sufficient to induce cell
activation. Other steps such as protein oligomerization or involvement
of another member of the LPS receptor complex may be required to induce
transmembrane signaling.
In this study we used HEK 293 cells since they do not express
endogenous TLR4, MD-2, or CD14. Through transient transfection we were
able to control expression levels of each of the three components of
LPS receptor, particularly of TLR4 that was reported to be expressed at
very low levels in most cells (11, 33). Furthermore, this cell system
allowed us to detect TLR4 and MD-2 as epitope-tagged proteins thereby
enriching their concentrations in immunoprecipitates and facilitating
detection by Western blotting. Thus the epitope-tagged proteins are
essential for detection of cross-linking. Previous studies failed to
demonstrate binding to proteins other than CD14 at the cell surface
presumably as a result of the low levels of surface expression of TLR4
and MD-2 (19, 34).
LPS binding to TLR4 and MD-2 readily occurred only in the presence of
the ternary complex composed of CD14, TLR4, and MD-2. As expected, CD14
represents the major binding site on the cell surface since it
contained more than 98% of total radioactive LPS bound to the cell.
Our data suggest that only a small fraction of the LPS bound to CD14 is
transferred to TLR4. This observation is reminiscent of the results of
Gegner et al. (31) who showed that most of the cellular
CD14-associated LPS appeared not to be involved in cellular activation.
Unfortunately the present system only provides qualitative binding data
and detailed analyses of affinities will require the development of
more precise analytical approaches.
The mechanism by which TLR4 transduces LPS signaling across the plasma
membrane is not known. Our observations suggest a complex mechanism for
activation of cells by LPS. LPS is transferred from a CD14·LBP
complex to a TLR4·MD-2 complex at cell surface. Several lines of
evidence support this model. First, LPS binds efficiently to TLR4 and
MD-2 only in the presence of CD14. Although MD-2 and TLR4 were both
strongly detected at cell surface of cells when co-expressed as shown
by FACS analysis, only faint binding of LPS to either protein could be
observed in the absence of CD14. CD14 can be present either on the cell
surface as a membrane-bound protein, or added to the cells as a soluble
form. Second, 28C5, an anti-CD14 antibody that has been shown to mask
the LPS-binding site on CD14, strongly abolishes LPS binding not only
to CD14, but also to TLR4 and MD-2. Thus there is a critical role of
CD14 in delivering LPS to TLR4 and MD-2. Finally, LPS binding to TLR4 and MD-2 was not affected in the presence of a mixture of metabolic inhibitors suggesting that transfer of LPS takes place at the plasma
membrane. The exact role of LPS binding to TLR4 and MD-2 with respect
to signal transduction is not yet clear. Surprisingly the anti-CD14
monoclonal 18E12 known to block LPS-induced cell activation without
preventing binding of LPS to CD14 (26), failed to prevent LPS binding
to TLR4 and MD-2. Moreover inhibitory anti-TLR4 monoclonal antibodies
had a limited or no effect of LPS binding to TLR4 or MD-2. One
interpretation of these data is that TLR4 has different domains in the
ectodomain; an LPS-binding site, an activation domain, and regions
involved in protein-protein interaction with TLR4 itself, MD-2 and/or
CD14. It is likely that oligomerization of TLR4 occurs following
interactions with LPS. Similarly, Poltorak et al. (33) have
also argued that LPS binds to TLR4 and initiates subsequent
oligomerization required for cell activation.
While biochemical and genetic studies establish a role of TLR2 as a
receptor for lipoproteins and peptidoglycan from diverse Gram-positive bacteria (28, 30, 35, 36) its role as a receptor for most
Gram-negative LPS under physiological conditions is in doubt (7, 11).
It does appear, however, that purified LPS is capable of activating
cells overexpressing TLR2 in the presence of MD-2 (12). Furthermore, it
has been shown that Previous studies suggested an essential role for MD-2 in determining
cellular responses to LPS (39). No data were provided to suggest a
mechanism for MD-2 function. Here we show that MD-2 is essential to
detect LPS binding to TLR4, a step that can be assumed to be essential
in the activation pathway. Information about a related protein MD-1 may
provide some insight into how MD-2 functions. MD-1 is a secreted
protein that binds to a protein known as RP105, a member of Toll
receptor family that is specifically expressed on B-lymphocytes (40,
41). MD-1 is thought to control expression of RP105 at the cell
surface. Shimazu and co-workers (8) have reported that MD-2 is
not expressed on the cell surface of the murine pro-B cell line Ba/F3
in the absence of TLR4. In contrast, our flow cytometry analyses showed
that MD-2 could be detected at the cell surface of 293 cells in the
absence of TLR4. TLR4 was also present, although weakly, at cell
surface when expressed alone. However, the presence of MD-2
dramatically enhanced cell surface-exposed TLR4. Alternatively MD-2 may
complex with TLR4 and enhance reactivity with the detecting antibodies.
However, since an identical enhancement was observed with anti-FLAG and HTA125, the anti-TLR4 monoclonal antibody, we feel the latter speculation is less likely. Preliminary studies in our laboratory have
shown that MD-2 may interact with selected regions of the ectodomain of
TLR4; studies are underway to define these interactions through
mutagenesis of TLR4. Preliminary experiments demonstrate that the first
N-terminal hundred amino acids of TLR4, containing 5 LRRs, play a
central role in binding LPS, since its deletion abolished LPS binding
but not association with MD-2 (data not shown).
The biochemical data we have presented together with results of others
(33) strongly supports a model where LPS binds directly to the known
proteins of the putative LPS receptor complex. This contrasts with what
is believed to occur in Drosophila where products of
microbial pathogens most likely do not interact directly with Toll-like
proteins. An essential question that remains unanswered is how binding
to TLR4 and MD-2 is linked to the generation of a transmembrane signal.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
LPS is cross-linked to TLR4 and MD-2 in a
CD14-dependent manner. Panel A, LPS
requires the presence of the tripartite complex composed of
CD14·TLR4·MD-2 to bind TLR4 and MD-2. 293 cells were co-transfected
with indicated plasmids (2.5 µg of FLAG-TLR4, 2.5 µg of FLAG-MD-2,
and 1 µg of CD14) and cultured for 2 days. Cells were incubated with
250 ng/ml 125I-ASD-Re595 LPS and 2.5 µg/ml LBP at
37 °C for 10 min, and photolyzed for 4 min on ice. Standard M2
precipitates of lysates were separated by SDS-PAGE, and analyzed by
autoradiography (top panel) or by immunoblotting
(bottom panel) using anti-FLAG antibody, indicating
expression of FLAG-TLR4 and FLAG-MD-2 proteins. Positions of molecular
mass standards (kDa) are shown on the left. Panel B, sCD14
can substitute mCD14 in transferring LPS to TLR4 and MD-2. Cells were
transfected with the indicated vectors and cultured for 2 days. Cells
were incubated with LBP and 125I-ASD-LPS plus 5 µg/ml
sCD14. Cells were photolyzed and immunoprecipitates analyzed as
described above.
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Fig. 2.
LPS specifically binds to TLR4 but not to
TLR1 and dToll. Panel A, LPS specifically binds to
TLR4. 293 cells were transfected with vectors encoding for CD14,
FLAG-MD-2 either in the presence of FLAG-TLR4, FLAG-TLR1, or
FLAG-dToll. Cells were harvested after 2 days and incubated with 2.5 µg/ml LBP and 250 ng/ml 125I-ASD-Re595 LPS at 37 °C
for 10 min. Cells were photolyzed and anti-FLAG immunoprecipitates
analyzed as described above. Panel B, cell surface
expression of TLR4, MD-2, TLR1, and dToll. 293 cells were transfected
with pEGFP-N3 vector and with specified plasmids for 2 days. Cells were
stained with anti-FLAG or HTA125 antibodies and analyzed with
FACSCalibur. Analysis gate was set on GFP positive cells. Experiments
were performed 3 times with similar results.
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Fig. 3.
LPS binds to TLR2. Panel A,
293 cells were transfected with the indicated plasmids (1 µg of CD14,
2.5 µg of FLAG-TLR2, and 2.5 µg of FLAG-MD-2) for 2 days. Cells
were washed and incubated with 2.5 µg/ml LBP and 250 ng/ml
125I-ASD-Re595 LPS, photolyzed, and anti-FLAG
immunoprecipitates analyzed as noted above. Panel B,
quantification of incorporated radioactivity to TLR2 by PhosphorImager
analysis. Panel C, cell surface expression of TLR2. 293 cells were transfected with TLR2 and analyzed with FACSCalibur as
described in the legend to Fig. 2.
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Fig. 4.
LPS binding to TLR4 and MD-2 occurs at cell
membrane. 293 cells were transfected with 2.5 µg of FLAG-TLR4 in
the presence of CD14 and FLAG-MD-2. Two days after transfection cells
were washed and preincubated in a buffer containing sodium azide,
sodium fluoride, and deoxyglucose for 10 min on ice. Cells were
incubated with 2.5 µg/ml LBP and 250 ng/ml 125I-ASD-Re595
LPS at 37 °C for 10 min. Cells were photolyzed and
immunoprecipitates analyzed as described above.
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[in a new window]
Fig. 5.
Specificity of LPS binding to TLR4 and
MD-2. Panel A, 293 cells were transfected with vectors
encoding for CD14, FLAG-TLR4, and FLAG-MD-2. Cells were harvested
48 h post-transfection and either left untreated or preincubated
with 100 µg/ml 0111:B4 LPS, 100 µg/ml 055:B5 LPS, 500 µg/ml LCD25
LPS, 5 µg/ml Lipid IVa, or 100 µg/ml lipoteichoic acid for 3 min at
37 °C prior to incubation with 250 ng/ml 125I-ASD-Re595
LPS and 2.5 µg/ml LBP. Panels B and C,
quantification of incorporated radioactivity to TLR4 and MD-2,
respectively, by PhosphorImager analysis.
View larger version (54K):
[in a new window]
Fig. 6.
28C5 blocks LPS binding to TLR4 and
MD-2. Panel A, 293 cells were transfected with vectors
encoding for CD14, FLAG-TLR4, and FLAG-MD-2. After 2 days, cells were
harvested and either left untreated or preincubated with 10 µg/ml of
each antibody 28C5, 63D3, or 18E12 for 1 h at 37 °C prior to
incubation with 250 ng/ml 125I-ASD-Re595 LPS and 2.5 µg/ml LBP. Panels B and C, quantification of
incorporated radioactivity to TLR4 and MD-2, respectively, by
PhosphorImager analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
T cells as well as macrophages derived from
C3H/HeJ mice respond to LPS from E. coli and
Porphyromonas gingivalis and lipid A
specifically through TLR2 (37, 38). Here we show that LPS and TLR2 are capable of interacting as evidenced by the finding of radiolabeled TLR2
following exposure to the derivatized Re595 LPS. However, in contrast
to our findings with TLR4, co-expression of MD-2 failed to influence
these interactions. It is important to emphasize that we could not
detect any cross-linked products when other members of the Toll family,
dToll or TLR1, were expressed at the surface of 293 cells even when
co-expressed with CD14 and MD-2. Thus we are confident that the
cross-linked products detected in cells expressing TLR4 or TLR2 reflect
specific LPS interactions with proteins functioning as receptors.
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ACKNOWLEDGEMENT |
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We thank Dr. Germana Sanna for careful reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI15136 (to R. J. U.), GM28485 (to R. J. U.), GM37696 (to R. J. U.), AI32021 (to P. S. T.), and HL23584 (to P. S. T.). This is Publication 136-IMM from The Scripps Research Institute, Department of Immunology.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.
¶ Supported by a fellowship of the Juvenile Diabetes Foundation.
To whom correspondence should be addressed. Tel.:
858-784-8219; Fax: 858-784-8239; E-mail: ulevitch@scripps.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M009164200
2 J. da Silva and R. J. Ulevitch, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; LBP, LPS-binding protein; 125I-ASD-LPS, radioiodinated sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Salmonella minnesota Re595 LPS; TLR, Toll-like receptor; dToll, Drosophila Toll; sCD14, soluble CD14; mCD14, membrane CD14; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorter.
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