From the Division of Inflammatory Diseases and
Synthetic Chemistry, Eisai Research Institute, Andover,
Massachusetts 01810 and the ¶ Boston University School of
Medicine, Boston Medical Center, The Maxwell Finland Laboratory for
Infectious Diseases, Boston, Massachusetts 02118
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ABSTRACT |
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TLR4 is a member of the recently identified
Toll-like receptor family of proteins and has been putatively
identified as Lps, the gene necessary for potent responses
to lipopolysaccharide in mammals. In order to determine whether TLR4 is
involved in lipopolysaccharide-induced activation of the nuclear
factor- Lipopolysaccharide
(LPS),1 a component of the
outer membrane of Gram-negative bacteria, is a potent activator of a
variety of mammalian cell types (1, 2). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in
approximately 20,000 annual deaths in the United States (3). Activation
of LPS-responsive cells, such as monocytes and macrophages, occurs
rapidly after LPS interacts with circulating LPS-binding protein and
CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein
necessary for sensitive responses to LPS (1, 2). LPS has been shown to
initiate multiple intracellular signaling events (4), including the
activation of NF- Toll is a transmembrane receptor in Drosophila that is
involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5, 6). Activation of the Toll receptor by
its ligand Spätzle results in the interaction and stimulation of
several signaling molecules that are homologous to proteins involved in
NF- Several lines of evidence suggest that one or more members of the TLR
family is the cell-surface receptor for LPS, the prototypical activator
of NF- Materials--
Human TLR4 cDNA was provided by Dr. Charles
A. Janeway, Jr. (Yale University). The ELAM-1-luciferase reporter
plasmid, pELAM-luc, was generated by cloning a fragment ( Cell Culture and Transfections--
HEK 293 cells (ATCC,
Rockville, MD) were cultured in Dulbecco's modified Eagle's medium
(ATCC, Rockville, MD) supplemented with 10% fetal bovine serum (Life
Technologies, Inc.). Cells were plated in 12-well tissue culture plates
(3 × 105 cells/well) and maintained in the above
medium for 24 h. Cells were transfected using the CalPhos
Maximizer protocol (CLONTECH) with 250 ng of TLR4
cDNA or vector DNA (pcDNA3, Invitrogen, Inc.) and 100 ng of
pELAM-luc. All cells were also transfected with a Purification of Soluble CD14 (sCD14) from Transfected CHO
Cells--
CHO cells expressing CD14 were cultured in suspension under
serum-free conditions (EX-CELL 301 medium supplemented with
L-glutamine). The culture supernatant was collected,
filtered through a 0.22-µm nitrocellulose filter, and concentrated
5-fold in a protein concentrator (Amicon Diaflo, PM30) with a 30-kDa
cut-off filter under pressure at 4 °C. This concentrate was then
loaded onto an anti-CD14 affinity column. The column was washed twice
with wash buffer before eluting with 0.1 M glycine, pH 2.8. The fractions were immediately neutralized with 1 M
Tris-HCl, pH 9.5, and an aliquot of each fraction was mixed with 2 × SDS loading buffer (Novex, San Diego, CA) and heated for 5 min at
95 °C. Expression and purification of sCD14 was verified by
SDS-polyacrylamide gel electrophoresis followed by silver stain and
immunoblotting with MEM-18 anti-CD14 antibody. Immunoreactive proteins
were visualized using enhanced chemiluminescence detection reagents
(Amersham Pharmacia Biotech).
Statistical Analysis--
Quantitative data are presented as
mean ± S.E. and analyzed using a statistical model based on a
one-way classification analysis of variance. Tests of significance for
all possible comparisons were determined by Student Newman-Keuls test
or unpaired t test (GraphPad Prism, version 2.0a).
To determine whether TLR4 mediates LPS-induced activation of
NF-B (NF-
B) pathway, HEK 293 cells were transiently
transfected with human TLR4 cDNA and an
NF-
B-dependent luciferase reporter plasmid followed by
stimulation with lipopolysaccharide/CD14 complexes. The results
demonstrate that lipopolysaccharide stimulates NF-
B-mediated gene
expression in cells transfected with the TLR4 gene in a dose- and
time-dependent fashion. Furthermore, E5531, a
lipopolysaccharide antagonist, blocked TLR4-mediated transgene
activation in a dose-dependent manner (IC50
~30 nM). These data demonstrate that TLR4 is involved in
lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-
B activation and
subsequent cellular events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, which ultimately leads to the synthesis and
release of a number of proinflammatory mediators, including
interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and
tumor necrosis factor-
(1). However, since CD14 is not a
transmembrane protein, it lacks the ability to transduce cytoplasmic
signals (2), and before the recent discovery of Toll-like receptors
(TLRs), the identity of a transmembrane protein that could relay
LPS-induced signals across the cell-surface membrane remained elusive.
B activation by the IL-1 receptor in mammalian cells (7, 8). The
cloning of a family of human receptors structurally related to
Drosophila Toll revealed five proteins that have
extracellular domains that contain multiple leucine-rich repeats and
cytoplasmic domains with sequence homology to the intracellular portion
of the IL-1 receptor (9). Furthermore, constitutively active mutants of
TLR2, TLR4, and TLR5 can induce the activation of NF-
B (10, 11), and
the active form of TLR4 increases the expression of NF-
B-regulated
genes for the inflammatory cytokines IL-1, IL-6, and IL-8 (11).
B and other proinflammatory responses. TLR2 and TLR4 are
highly expressed in cells that respond to LPS, such as peripheral blood
leukocytes, macrophages, and monocytes (11, 12). Also, heterologously
expressed TLR2 mediates LPS-induced NF-
B activation and IL-8
mRNA expression in HEK 293 cells (12, 13). However, TLR2 is not the
only potential LPS signal transducer. The C3H/HeJ mouse is a
spontaneous LPS resistant mutant. Poltorak et al. (14)
mapped the Lps gene, which has been shown previously to be
necessary for LPS responses in LPS nonresponder C3H/HeJ mice, to TLR4.
TLR4 from the C3H/HeJ mouse has a single point mutation at amino acid
712 (Pro to His) that changes the function of the receptor dramatically
(14); furthermore, the LPS-resistant C57/10ScCr mouse appears to be
null for the TLR4 locus. These observations strongly support the
concept that TLR4, and not TLR2, is the dominant LPS receptor in
mammals and the hypothesis that TLR4 is a cell-surface component of the
LPS signaling pathway. Thus, the present study was conducted to
investigate whether TLR4 is involved in mediating the actions of
LPS.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
241 to
54
base pairs) of the human E-selectin promoter into the pGL3 reporter
plasmid (Promega, Inc.). All plasmid constructs were confirmed by
automated sequencing analysis. Lipopolysaccharide was purchased from
Sigma. The human embryonic kidney cell line HEK 293 (CRL-1573) was from
American Type Culture Collection (Rockville, MD). The Chinese hamster
ovary (CHO) cell line expressing CD14 was engineered and maintained as
described (15). The LPS antagonist (E5531) was synthesized as described
previously (16). Plasmid DNA was isolated with Qiagen
Endo-freeTM Maxi-prep columns (Chatsworth, CA). MEM-18
anti-CD14 antibody was purchased from Accurate Chemicals & Scientific
Corp. (Westbury, NY). Phorbol 12-myristate 13-acetate (PMA) and IL-1
were purchased from Calbiochem and Endogen (Woburn, MA), respectively.
Luciferase activity was assayed using a commercial luciferase assay kit
(Stratagene, La Jolla, CA).
-galactosidase
control plasmid for normalizing transfection efficiencies. After
transfection, cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 1% fetal bovine serum overnight (18 h). The
following day, cells were either left untreated or incubated with the
indicated amount of ligand and/or compound E5531. After the indicated
treatment period, cells were harvested in lysis buffer and assayed for
luciferase activity per the manufacturer's protocol. The amount of
luciferase activity in each sample was quantified by a Wallac 1450 MicroBetaTrilux counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, HEK 293 cells were transiently transfected with TLR4 cDNA or empty vector control (pcDNA3) and an
NF-
B-dependent ELAM-1-luciferase reporter plasmid
(pELAM-luc). Twenty-four hours post-transfection, cells were left
untreated or incubated with either LPS (1 µg/ml or indicated
concentrations), sCD14 (10 nM) or both for an additional 6 h. Cells were then lysed and assayed for luciferase activity. Expression of TLR4 in HEK 293 cells induced activation of the NF-
B
reporter gene 2.5-fold above controls (cells transfected with empty
vector and the pELAM-luc reporter gene) in the absence of stimuli (Fig.
1). Soluble CD14 alone did not have a
significant effect on NF-
B activity in the presence or absence of
TLR4. LPS treatment alone (1 µg/ml) was sufficient to elicit an
increase (1.6-fold) in luciferase activity after incubation with TLR4
transfected cells, and this increase was not observed in vector
controls (Fig. 1). However, when cells were stimulated with LPS in the
presence of sCD14, there was a marked increase in TLR4-mediated
activation of NF-
B that was not observed in cells treated with LPS
or CD14 alone (Fig. 1). Stimulation of TLR4-mediated NF-
B activation by LPS plus CD14 was 5-fold higher than levels produced by TLR4 expression in unstimulated controls or with CD14 alone. LPS-induced reporter activity occurred in a dose-dependent fashion,
increasing 2-fold above controls at 10 ng/ml LPS and reaching maximal
levels at 1 µg/ml LPS (Fig.
2A). Furthermore, stimulation
of NF-
B activity by LPS was time-dependent and reached
maximal levels at 18-24 h after LPS addition (Fig. 2B).
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Fig. 1.
LPS increases TLR4-mediated
NF- B activation via a CD-14 dependent
mechanism. HEK 293 cells were plated at a density of 3 × 105 cells/well in 12-well plates and maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
for 24 h. Cells were transiently transfected with TLR4 cDNA or
vector DNA and the ELAM-1-luciferase reporter plasmid as described
under "Experimental Procedures." Cells were either left untreated
or stimulated with CD14 (10 nM), LPS (1 µg/ml), or the
combination of CD14 plus LPS for 6 h. After cell lysis, luciferase
activity was as described under "Experimental Procedures." These
data represent the mean ± S.E. from seven independent
experiments. Means with different superscripts are significantly
different from one another, p < 0.05.
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[in a new window]
Fig. 2.
LPS increases TLR4-mediated
NF- B activation in a dose- and
time-dependent manner. HEK 293 cells were transiently
transfected with TLR4 cDNA or vector DNA and pELAM-luc. Cells were
either left untreated or exposed to sCD14 (10 nM) and the
indicated concentration of LPS for 6 h (A) or 1 µg of
LPS/ml for the indicated amount of time (B) as described in
the legend to Fig. 1. After cell lysis, the amount of luciferase
activity in each sample was quantified. These data represent the
mean ± S.E. from three independent experiments. Means with
different superscripts are significantly different from one another,
p < 0.05.
In order to further examine whether TLR4 is involved in LPS
signaling at the cell surface, HEK 293 cells were transfected with TLR4
or empty vector and the NF-B reporter plasmid. Subsequently, cells
were co-incubated with LPS (plus CD14) and increasing concentrations of
E5531, an LPS antagonist that has been shown to inhibit LPS-induced cytokine synthesis in macrophages and in vivo (16).
Measurements of cellular luciferase activity after these treatments
indicated that E5531 inhibits TLR4-mediated NF-
B activation in a
dose-dependent manner (Fig.
3A). At 10 nM,
E5531 significantly reduced NF-
B-dependent gene
activation by LPS, and at higher drug concentrations (1 and 10 µM), NF-
B reporter activities in TLR4 expressing cells
were similar to unstimulated controls. In contrast to LPS-stimulated TLR4-expressing cells, 1 µM E5531 did not affect PMA- or
IL-1
-induced NF-
B gene activation in these cells (Fig.
3B), indicating that the drug selectively inhibited
TLR4-mediated NF-
B activation in response to LPS.
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DISCUSSION |
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Although it has been proposed previously that LPS interacts
with a transmembrane receptor or recognition molecules on the surface
of plasma membranes of responsive cell types (2), strong evidence for
this hypothesis has only recently become available. First, Medzhitov
et al. (11) identified a human homolog of
Drosophila Toll, later designated TLR4 (9), with signaling
properties similar to those observed for the IL-1 receptor. These
properties include the activation of the transcription factor NF-B
and induction of mRNA for several proinflammatory cytokines (11),
both of which were also observed when responsive cells are activated
with LPS (1). Second, reports by Yang et al. (12) and
Kirschning et al. (13) demonstrate the ability of TLR2 to
signal in the presence of LPS and CD14, strongly suggesting a role for
this protein in LPS action. Kirschning et al. (13) also
reported that TLR1 and TLR4 failed to increase NF-
B reporter
activity in the presence of LPS, further supporting the notion that
TLR2 is a specific component of the cellular receptor for LPS. However, the recent report by Poltorak et al. (14), which identifies the genetic lesion in the LPS-resistant C3H/HeJ mice as a mutation in
the tlr4 gene (14), prompted us to examine the role of TLR4 in LPS signaling.
In the experiments presented here, transfection of HEK 293 cells using
a construct containing the full-length cDNA for TLR4 was sufficient
to elicit a significant response with LPS in the presence of soluble
CD14. The reporter construct utilized in these experiments contained a
region of the promoter for the E-selectin gene, which is absolutely
dependent upon NF-B activation for activity (17). Thus, LPS-induced
stimulation of our ELAM-1 reporter gene in TLR4 expressing cells is
predominantly mediated via the NF-
B signaling pathway. Previous
studies have shown that constitutively active TLR4 constructs can
activate proximal components (MyD88, IL-1 receptor-associated kinase,
tumor necrosis factor receptor-associated factor-6, and NF-
B
inducing kinase) of the IL-1 signaling pathway that lead to NF-
B
activation (18, 19). Although we demonstrate a link between LPS
signaling and TLR4 expression in this study, it remains to be
determined whether the effects of LPS utilize the same subsequent
signaling proteins as the IL-1 receptor (4).
Kirschning et al. (13) failed to observe LPS-inducible NF-kB
activation in HEK 293 cells transfected with human TLR4. The reasons
for this are unclear, but might reflect the differences inherent in HEK
293 cell lines. For example, we have found that one stock of HEK 293 cells responded to LPS, as evidenced by inducible NF-B
translocation, in the absence of transfection. In order to perform
these studies, we were forced to locate an alternative stock of HEK 293 cells, as this particular lot, acquired directly from the distributor
(ATCC, Rockville, MD), expressed high levels of TLR2
mRNA.2 Like the IL-1
receptor, TLRs might require the formation of heterodimeric signaling
complexes with highly homologous proteins (such as another TLR) upon
ligand binding (20). A testable hypothesis that might explain the
differences in outcome between seemingly identical experiments is that
the background expression of TLRs determines which heterodimers can be
formed after gene transfer and that all strains of HEK 293 cells are
not equivalent in this respect. Other experimental differences might
also affect results obtained in these types of experiments, such as the
nature of the TLR4 construct, the amount of DNA utilized in
transfections, or other conditions that may influence TLR4 expression.
For example, we have shown that the amount of TLR4 expression vector
transfected into cells influences the regulation of pELAM-luc activity
by LPS.3 This was presumably
due to elevated basal-specific activity of pELAM-luc that was increased
when higher amounts of TLR4 were expressed. A similar effect has been
reported in HEK 293 cells expressing I
B kinase-
(21). In support
of Yang et al. (12) and Kirschning et al. (13),
we have shown that our HEK 293 cells transfected with a construct
containing TLR2 are also responsive to LPS plus
CD14.3
E5531 is a potent synthetic lipid A analog that acts as an
antagonist of LPS-induced activation (16). The compound inhibits the
effects of LPS in monocytes, macrophages, animal models of sepsis and
infection (16), as well as the effects of low amounts of LPS
administered to humans (22). Based on LPS binding studies, it is
believed that E5531 antagonizes LPS activity at its cell-surface receptor, leading to inhibition of transmembrane signal transduction (16). Consistent with this hypothesis, E5531 inhibits the stimulation of TLR4 transfected cells by LPS plus CD14 in a
dose-dependent manner with an IC50 of ~30
nM, comparable with its effects in other cell based assays
(23). Since E5531 did not affect activation of the NF-B reporter
gene induced by IL-1
or the actions of interferon-
in murine
macrophages (16), this indicates that the inhibitory activity of E5531
is closely linked to and specific to cell-surface components utilized
by LPS. In these experiments, TLR4 is the only protein whose expression
can account for the LPS responsiveness observed in transfected cells.
Therefore, based on these results, it is very likely that TLR4 is a
receptor for LPS and that E5531 acts as an antagonist of this interaction.
In light of the recent identification of the lesion in C3H/HeJ and
C57BL/10ScCr LPS-resistant mice as a mutation in the tlr4 gene (14), the results presented here are particularly significant. These data demonstrate that TLR4 behaves as a functional LPS receptor when transfected into cells that are otherwise LPS-insensitive. This
cell-based result is consistent with the observation made by Poltorak
et al. (14) in C3H/HeJ mice. These data do not exclude a
role for TLR2 in LPS signal transduction under certain conditions or in
specific cell types, but support the hypothesis that TLR4 is essential
in LPS signaling in vitro and in vivo. Finally, the characterization of TLR4 as a receptor for the LPS antagonist is an
important development for understanding the mechanistic steps and will
enable further improvements in the discovery of anti-endotoxin agents.
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ACKNOWLEDGEMENTS |
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We thank Hua Yang and Melissa Ferrin for excellent technical assistance and Samantha Roberts for assisting in the preparation of the manuscript.
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FOOTNOTES |
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* 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: Eisai Research Institute, 100 Research Dr., Wilmington, MA 01887. Tel.: 978-661-7276; Fax: 978-657-7715.
** Supported by National Institutes of Health Grant RO1 GM54060.
2 E. Lien and D. T. Golenbock, unpublished observations.
3 J. C. Chow and F. Gusovsky, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
LPS, lipopolysaccharide;
TLR, toll-like receptor;
IL, interleukin;
NF-B, nuclear factor-
B;
sCD14, soluble CD14;
CHO, Chinese hamster ovary;
PMA, phorbol 12-myristate 13-acetate;
pELAM-luc, ELAM-1-luciferase
reporter plasmid.
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