From Tularik Inc., South San Francisco, California
94080 and the § Northwest Center for Medical Education,
Indiana University School of Medicine, Gary, Indiana 46408-1197
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ABSTRACT |
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The life-threatening complications of sepsis in
humans are elicited by infection with Gram-negative as well as
Gram-positive bacteria. Recently, lipopolysaccharide (LPS), a major
biologically active agent of Gram-negative bacteria, was shown to
mediate cellular activation by a member of the human Toll-like receptor
family, Toll-like receptor (TLR) 2. Here we investigate the mechanism of cellular activation by soluble peptidoglycan (sPGN) and lipoteichoic acid (LTA), main stimulatory components of Gram-positive bacteria. Like
LPS, sPGN and LTA bind to the glycosylphosphatidylinositol-anchored membrane protein CD14 and induce activation of the transcription factor
NF- Infection with bacteria may cause sepsis and lead to septic shock,
characterized by refractory hypotension and eventually multiorgan
failure and death (1-3). Gram-positive sepsis has been recognized as
an important clinical condition (4-6). Its causative agents are cell
wall components of Gram-positive bacteria, like peptidoglycan
(PGN)1 and lipoteichoic acid
(LTA). PGN, which predominates the cell wall of Gram-positive bacteria,
is an alternating The initial event in LPS recognition is its complexation with
LPS-binding protein (LBP) in the plasma. This LPS·LBP complex subsequently binds to CD14, which triggers cell activation (1, 11).
Because anti-CD14 monoclonal antibodies and LPS antagonists were found
to inhibit activation of CD14 positive monocytes by PGN and LPS, CD14
was proposed to be the receptor for both PGN and LPS (8). Consequently,
it has been demonstrated that the binding of PGN (12, 13) and LTA (9,
10) to CD14 lead to CD14-dependent cell activation.
Because CD14 is present either as a
glycosylphosphatidylinositol-linked membrane receptor lacking an
intracellular signaling domain or in soluble form in serum (14), a
transmembrane co-receptor has been postulated in LPS responsive cells,
which transduces the LPS signal into the cytoplasm (1). Recently, two
candidate signal transducing receptors have been identified which are
members of the Toll-like receptor (TLR) family, TLR2 and TLR4
(15-19).
Toll controls dorsoventral patterning during embryogenesis in
Drosophila melanogaster (20) and is involved in the host
defense reactions upon fungal infection in the adult fly (21). Data base searches have identified a family of human Toll homologues, termed
Toll-like receptors (22-25). The intracellular domain of TLRs
resembles that of the mammalian IL-1 receptor (IL-1R), thereby defining
the Toll/IL-1R receptor family. Consistent with this, Drosophila Toll signaling is similar to mammalian IL-1R
signaling (26). Several members of the Toll/IL-1 receptor family have been shown to activate NF- Because Drosophila Toll participates in innate immune
responses in flies, previous studies examined whether members of this family are involved in these responses in mammalian systems. These studies implicated TLR family members TLR2 and TLR4 as mediators of
responses to LPS (15-17, 19). In this study we demonstrate that PGN
and LTA from Gram-positive bacteria can activate cells in a
TLR2-dependent manner.
Cell Culture and Biological Agents--
A subclone of the human
embryonic kidney HEK293 cell line has been maintained at Tularik, Inc.,
since 1993 as described (28). Tissue culture experiments were performed
in the presence of 10% certified fetal bovine serum (Life
Technologies, Inc.) unless stated otherwise. The serum was not
heat-inactivated and contained endotoxin at <10 enzyme units/ml as
specified by the manufacturer. Purified preparations of soluble PGN
(sPGN) from Staphylococcus aureus Rb were used to stimulate
cultured cells (4). sPGN was isolated by vancomycin affinity
chromatography and the purity of sPGN was established by quantitative
chemical analysis, silver stain analysis after polyacrylamide gel
electrophoresis, and fluoroautoradiography of sPGN biosynthetically
labeled with [14C]alanine. The purity of the sPGN was
estimated at Expression Plasmids--
Expression plasmids for CD14, TLRs, and
the TLR truncation mutant were generated as described earlier (15).
NF- LPS, a component of the outer membrane of the envelope of
Gram-negative bacteria, is capable of eliciting immune stimulatory effects similar to whole bacteria in the host organism. Activation of
NF-B in host cells like macrophages. We show that whole Gram-positive bacteria, sPGN and LTA induce the activation of NF-
B
in HEK293 cells expressing TLR2 but not in cells expressing TLR1 or
TLR4. The sPGN- and LTA-induced NF-
B activation was not inhibited by
polymyxin B, an antibiotic that binds and neutralizes LPS. Coexpression
together with membrane CD14 enhances sPGN signal transmission through
TLR2. In contrast to LPS signaling, activation of TLR2 by sPGN and LTA
does not require serum. These findings identify TLR2 as a signal
transducer for sPGN and LTA in addition to LPS.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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(1, 4) linked N-acetylmuramyl and
N-acetylglucosaminyl glycan whose residues are cross-linked
by short peptides. LTA is anchored in the membrane by glycolipids found
in many Gram-positive bacteria (7). Like lipopolysaccharide (LPS), a
component of the outer cell membrane of Gram-negative bacteria,
isolated PGN and LTA can elicit most of the clinical manifestation of
bacterial infection. These bacterial agents stimulate the excessive
release of pro-inflammatory cytokines like tumor necrosis factor, IL-1,
and IL-6, and other inflammatory mediators from immune cells, including
macrophages, which ultimately cause clinical symptoms (8-10).
B (15, 19, 22, 24, 27). Overexpression of
a constitutively active mutant of TLR4 (also termed human Toll) has
been shown to induce the activation of NF-
B in mammalian cells,
leading to induction of NF-
B-dependent genes including those encoding inflammatory cytokines and the T cell costimulatory protein B7.1 (24). Although the extracellular domains of the IL-1
receptors identify them as members of the IgG superfamily, the
extracellular domains of human Toll-like receptors and
Drosophila Toll lack IgG domains and share characteristic
repeated leucine-rich motives (25).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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98.5%. The average molecular mass was 125,000 Da (4,
29, 30). PGN from Streptococcus pyogenes Group A was
purchased from Lee Chemicals Inc. (Grayson, GA). LTA preparations from
Bacillus subtilis, ATCC 6051, S. pyogenes, strain
IID 698, and Streptococcus sanguis, Type I, ATCC10556 were
purchased from Sigma. All LTAs applied were prepared using a phenolic
extraction procedure (31) and tested for LPS contamination by
incubation with polymyxin B sulfate (Calbiochem-Novabiochem Co., La
Jolla, CA). All different PGN and LTA preparations behaved similarly in
cell activation experiments. For control stimulation, LPS from
Escherichia coli serotype 0111:B4 prepared by phenol
extraction (Sigma) was used. Gram-positive bacteria of the species
S. aureus ATCC 29213 and B. subtilis pY79 (prototroph SP
s) (kindly provided by Dr. Simon Lynch, Tularik Inc.)
were plated on Penassay broth agar culture plates, transferred to Luria
broth, and cultured by shaking at 37 °C overnight. Bacteria were
pelleted and washed three times with ice-cold phosphate-buffered saline. Aliquots of 15-fold concentrated bacterial suspensions were
frozen at
80 °C. The cell titer of bacterial suspensions was
6 × 1010 cells/ml for S. aureus and 9 × 109 cells/ml for B. subtilis.
B Reporter Assays--
Human embryonic kidney HEK293 cells
(3 × 105) were plated in 3.5-cm dishes and
transfected on the following day by the calcium phosphate precipitation
method (32) with the indicated amounts of expression vectors, 0.1 µg
of ELAM-1 luciferase reporter plasmid (33), and 1 µg of RSV
-galactosidase plasmid for normalization. The cells were lysed, and
luciferase activity was determined using protocols and reagents from
Promega Co. (Madison, WI). Luciferase activities were calculated as
fold induction compared with unstimulated vector control. All bar
diagrams are shown as the means ± S.E. for one representative
experiment in which each transfection was performed in duplicate. Each
experiment was repeated at least twice in all cases.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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B by LPS in mammalian HEK293 was shown to depend on the expression of TLR2. This finding prompted us to investigate whether Gram-positive bacteria and their immunologically active components PGN
and LTA utilize members of the TLR family to signal cellular activation. Human embryonic kidney HEK293 cells were transiently transfected with a NF-
B-dependent E-selectin promoter
luciferase reporter gene. NF-
B-driven reporter gene activation was
observed in cells stimulated with whole Gram-positive bacteria of the
species S. aureus and B. subtilis in cells
expressing TLR2 but not in cells expressing TLR1 or TLR4 (Fig.
1). To investigate whether PGN and LTA
were responsible for NF-
B activation induced by whole Gram-positive
bacteria, purified sPGN and LTA were used for subsequent experiments.
As previously observed for LPS, no significant induction of reporter
gene activity was detected after stimulation with sPGN or LTA in cells
transfected with control vector alone (Fig. 2). In addition, expression of CD14 did
not mediate responsiveness to sPGN and LTA in the absence of TLRs (Fig.
2). Transient overexpression of TLR4 constitutively activated the
reporter gene, and no significant increase in activity was observed
upon stimulation with sPGN or LTA (Fig. 2). Overexpression of TLR1 and
TLR2 caused only marginal reporter gene activation, which was strongly
induced by stimulation with sPGN or LTA only in cells expressing TLR2
but not TLR1 (Fig. 2). Again, this is similar to results obtained for
LPS stimulation (15). Cells expressing a TLR2 mutant lacking the
intracellular domain (TLR2 1-720) did not respond to sPGN or
LTA (Fig. 2). Thus, the intracellular domain of TLR2 is essential for
signal transduction.
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Fig. 1.
Effect of TLRs on
NF- B activation by whole Gram-positive
bacteria. Luciferase activities were determined in 3 × 105 HEK293 cells plated in 3.5-cm dishes and transfected
with 0.2 µg of vector control (open bars), TLR1
(stippled bars), TLR2 (closed bars), or TLR4
(hatched bars) together with 0.1 µg of ELAM-1 luciferase
reporter plasmid and 1 µg of RSV
-galactosidase plasmid 24 h
after transfection. Cells were either left untreated or stimulated with
20 µl of bacterial suspension/3.5-cm dish of S. aureus
(6 × 1010 cells/ml) or B. subtilis (9 × 109 cells/ml) as indicated.
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Fig. 2.
Effect of mCD14 and TLRs on PGN- and
LTA-induced NF- B activation. Luciferase
activities were determined in 3 × 105 HEK293 cells
plated in 3.5-cm dishes and transfected with the indicated expression
plasmids (0.2 µg) together with ELAM-1 luciferase reporter plasmid
(0.1 µg) and 1 µg of RSV
-galactosidase plasmid 24 h after
transfection. Cells were either left untreated (open bars)
or stimulated with PGN (10 µg/ml; closed bars) or LTA (10 µg/ml; hatched bars) for 6 h before harvest.
sPGN and LTA induced NF-B-dependent reporter gene
activation in TLR2-expressing cells occurred in a
dose-dependent manner (Fig.
3). Coexpression of CD14 with TLR2 led to
a slight increase in reporter gene activation upon sPGN stimulation.
The effect was more pronounced at lower sPGN concentrations (Fig.
3B). Coexpression of CD14 had no significant effect on LTA
stimulation (Fig. 3C). Cellular activation by PGN is
CD14-dependent like activation by LPS, whereas LTA
induction might be CD14-independent. On the other hand, dependence of
cell activation by PGN and LTA on CD14 has been shown for hematopoietic
cells on the basis of genetic evidence and CD14 blocking experiments
(8-10). HEK293 cells might lack a component of a receptor complex
required for full CD14 function. Alternatively the model system in
HEK293 cells may resemble a receptor complex for detection of microbial
components by cells of nonhematopoietic origin. Although the molecular
basis for responsiveness to microbial agents may be similar in both
cell types (34), differences in recruitment of CD14 and other
components may exist for specific stimuli.
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It has been demonstrated that LPS signaling is dependent on serum as a
source for LBP and/or soluble CD14 (sCD14). Consistent with this, LPS
signaling via TLR2 in HEK293 cells is serum-dependent (15,
19) (Fig. 4A). In contrast,
removal of serum before stimulation with sPGN or LTA did not inhibit
reporter gene activation (Fig. 4, B and C). Under
serum-free conditions sPGN and LTA stimulation were even markedly
increased compared with serum-containing conditions (Fig. 4,
B and C). Serum components might compete for sPGN
and LTA binding with cellular receptors and thereby neutralize
immunostimulatory activity.
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In Drosophila, genes encoding antimicrobial peptides are
activated by Toll upon fungal infection (21), whereas the Toll homologue 18-wheeler regulates antimicrobial peptide synthesis in
response to bacterial infection (35). Evidence for the existence of
mammalian signal transducers with similar function was reported (1).
The present study suggests the involvement of TLR2 in the activation of
NF-B by Gram-positive bacteria based on overexpression of TLRs in
mammalian HEK293 cells. Our results indicate that TLR2 may not be
exclusively a pattern recognition receptor for LPS from Gram-negative
bacteria as proposed by others (19) but may also act as a receptor for
PGN and LTA from Gram-positive bacteria. This does not exclude
differential composition of a receptor complex upon stimulation with
different microbial components. Genetic evidence from two different
mouse strains, LPS hyporesponsive C3H/HeJ and C57BL/10ScCr mice, has
recently implicated TLR4 (human Toll) to be crucial for immune
responses toward Gram-negative infection (16, 17). Interestingly,
differences in the cellular response to LPS and components of
Gram-positive bacteria were reported for one of these mouse strains.
C3H/HeJ mice harbor a single inactivating point mutation of TLR4, which
results in hyporesponsiveness to LPS. However, C3H/HeJ mice respond
normally to stimulation with preparations from Gram-positive group B
streptococci type III as evaluated by NF-
B activation and tumor
necrosis factor release (36). Thus TLR4 may not be involved in the
cellular responses to Gram-positive bacteria. On the other hand,
C57BL/10ScCr mice, which lack TLR4 expression, are impaired in
interferon-
release upon stimulation with a variety of microbial
agents, whereas C3H/HeJ mice are not (37). Reduced responsiveness to
bacterial dextran B512 in C57BL/10ScCr mice may be due to an additional genetic defect as indicated earlier (38). Therefore the contribution of
individual members of the TLR family to signaling by bacterial or other
microbial stimuli in vivo remains to be elucidated.
Nevertheless, the implication of TLR2 in detection and signal
transduction of PGN and LTA by host cells provides further insight into
the mechanisms of innate immune response to Gram-positive infection.
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ACKNOWLEDGEMENTS |
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We thank Simon Lynch for providing bacteria and Zhaodan Cao and Art Okamoto for helpful discussions.
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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.
¶ Supported by National Institutes of Health Grant AI28797.
Present address: Institut fuer Med. Mikrobiologie, Immunologie
und Hygiene, Trogerstrasse 4b, 81675 Muenchen, Germany.
** To whom correspondence should be addressed. Present address: DeveloGen AG, Rudolf-Wissel-Strasse 28, 37079 Goettingen, Germany.
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ABBREVIATIONS |
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The abbreviations used are: PGN, peptidoglycan; sPGN, soluble PGN; LTA, lipoteichoic acid; LPS, lipopolysaccharide; TLR, Toll-like receptor; IL, interleukin; IL-1R, IL-1 receptor; LBP, LPS-binding protein; RSV, Rous sarcoma virus.
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