From the Institut für Mikrobiologie und
Hygiene, Charité Medical Center, Humboldt-University, 10117 Berlin, the § Department of Biochemical Pharmacology,
University of Konstanz, 78457 Konstanz, the ¶ Institute for
Microbiology, Immunology and Hygiene, Technical University, 81657 Munich, and the
Department of Immunochemistry, Forschungszentrum
Borstel, 23845 Borstel, Germany
Received for publication, November 20, 2000, and in revised form, March 30, 2001
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ABSTRACT |
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Recently Toll-like receptors (TLRs) have been
found to be involved in cellular activation by microbial products,
including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4,
TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of
recognition of lipoteichoic acids (LTAs) and related glycolipids has
not been completely understood. In order to determine the role of TLRs
in immune cell activation by these stimuli, experiments involving
TLR-2-negative cell lines, TLR-expression plasmids, macrophages from
TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were
performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as
highly purified LTAs from Staphylococcus aureus and
Bacillus subtilis exhibited TLR-2 dependence in nuclear
factor Translocation of nuclear factor Signaling pathways leading to NF- Cellular activation by microbial ligands via members of the family of
Toll-like receptors (TLRs) has recently been found to initiate a
signaling cascade also resulting in translocation of NF- Lipoteichoic acids (LTAs) are present in the cell wall of most
Gram-positive bacteria and are linked to the cytoplasmic membrane (42).
Most widespread are poly(glycerophosphate) LTAs such as the ones found
in Staphylococcus aureus and Bacillus subtilis (43, 44). They are macroamphiphiles commonly consisting of hydrophilic
polyglycerol phosphate chains and a diacylglycerol lipid anchor (45).
LTAs exhibit immunostimulatory activity such as cytokine induction (46,
47). It thus was tempting to speculate that LTAs also utilize TLRs for
activation of immune cells. However, results obtained in different cell
systems have been controversial, and the role of TLRs in LTA-induced
cell stimulation has not been completely understood; S. aureus and Streptococcus sanguis LTA induced IL-6 and
nitric oxide in macrophages from wild-type and TLR-2-deficient mice,
but not in macrophages from TLR-4-deficient mice (48). In contrast,
overexpression of TLR-2 in HEK293 cells conferred inducibility of
NF- Previously we have reported on the isolation of two novel
Treponema species, T. maltophilum, associated
with peridontitis in humans, and T. brennaborense, found in
bovine cattle disease (50-52). Isolated membrane glycolipids of both
treponemes, which share structural characteristics with LTAs, stimulate
mononuclear cells to release TNF- In order to identify the TLRs involved in stimulation of immune cells
by Treponema glycolipids and LTAs, these two different glycolipids, highly purified butanol-extracted LTAs from B. subtilis and S. aureus, and, as control, LPS were used
for stimulation of cells differing in their TLR expression pattern. Our
results confirm our previous findings and indicate that NF- Treponema Culture and Preparation of Phenol/Water
Extracts--
Frozen stocks of T. brennaborense and T. maltophilum cells (300 µl, each stored at
For glycolipid extraction, aqueous suspensions of treponeme cells were
digested with RNase (Sigma, Deisenhofen, Germany), DNase (Merck,
Darmstadt, Germany), and proteinase K (Merck). The suspensions were
dialyzed and extracted using a hot phenol/water extraction method (54).
In brief, the phenol/water extraction was performed by mixing the cell
suspension with an equal volume of 90% phenol and stirring at 68 °C
for 10 min. After cooling on ice, the mixture was centrifuged at
3,000 × g for 10 min at 0 °C, and the upper phase
was collected. This procedure was repeated twice, and combined phases
were dialyzed and lyophilized. LPS contamination was thoroughly
investigated by Limulus amoebocyte lysate assay and other
methods and could be clearly ruled out. This has been published
elsewhere (53). A mock extract including all media and chemicals that
were used during extraction was tested in all assays.
Extraction of LTAs from B. subtilis and S. aureus--
B.
subtilis (DSMZ 1087) and S. aureus (DSM20233) were
grown in a 8-liter shaker and a 35-liter fermentor, respectively. After harvesting by centrifugation at 4 °C and 8000 rpm, the pelleted bacteria were sonicated (Branson Sonifier, Branson,
Schwäbisch-Gemünd, Germany) on ice and extracted with
butanol at room temperature (49). The aqueous phase was purified by
hydrophobic interaction chromatography (HIC) on octyl-Sepharose.
Fractions were screened for phosphorus-rich LTA by a phosphomolybdene
blue assay (55); positive fractions were pooled. All LTA-preparations
were negative in a chromogenic Limulus amoebocyte lysate
assay (QCL-1000, Bio Whittaker, Walkersville, MD), i.e. they
contained less than 30 pg of LPS/mg of LTA from B. subtilis
and less than 6 pg of LPS/mg of LTA from S. aureus. The
purity of the preparations was estimated by NMR analysis to be
Stimulation of the Murine Macrophage Cell Line RAW264.7 and
Chinese Hamster Ovary (CHO) Cells and Estimation of NF-
CHO cells transfected with human CD14 (generously provided by L. Hamann, Forschungszentrum Borstel, Borstel, Germany) were cultured with
Ham's nutrient medium F-12 (PAA Laboratories GmbH, Linz, Austria)
supplemented with 10% FCS and 400 µg/ml hygromycin B (Calbiochem,
San Diego, CA). 4 × 105 CHO/CD14 cells were plated in
six-well tissue culture plates. At the next morning, cells were starved
in FCS-free Ham's medium for 3 h before stimulation with
Treponema glycolipids, LTAs, or E. coli 0111:B4
LPS (Sigma) in the presence of 2% non-heat-inactivated FCS.
After 1 h cells were washed with ice-cold phosphate-buffered
saline containing 1 mM Na3VO4, and
incubated in 150 µl of buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1 mM
Na3VO4, 0.5 mM phenylmethylsulfonyl
fluoride, 1 mM leupeptin, and 1 mM NaF). After
20 min, cells were harvested mechanically, transferred to 1.5-ml tubes,
mixed with 25 µl of Nonidet P-40, and centrifuged at 13,000 × g at 4 °C for 1 min. Pellets were resuspended in 50 µl
of buffer B (400 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
leupeptin, and 1 mM NaF), incubated for 30 min at 4 °C,
spun at 13.000 × g at 4 °C for 5 min, and
supernatants containing nuclear proteins were collected. The binding
activity of NF- Stimulation of RAW264.7 and U373MG Cells and Detection of
Cytokine Concentration--
To assess induction of TNF-
For measurement of human IL-6, 3.75 × 104 cells/well
of U373MG cells (33, 56) were seeded in 96-well tissue culture plates with DMEM containing 10% FCS. In certain experiments, U373MG cells were incubated with the anti-TLR-4 antibody HTA125 (57) at
concentrations of 5 µg/ml for 1 h prior to stimulation. On the
next day, the cells were incubated overnight with Treponema
glycolipids, LTAs, or E. coli 0111:B4 LPS (Sigma) in the
presence of 5% AB serum. MaxiSorp ELISA plates were incubated with 3 µg/ml anti-hIL-6 capture Ab (R&D) in 100 mM
Na2CO3, pH 8.3. Samples and IL-6 standard (R&D Systems) were incubated at room temperature for 2 h, detection was
performed employing 25 ng/ml biotinylated anti hIL-6 Ab (R&D Systems),
and substrate reaction was carried out as in the TNF- Fractionation of Treponema Extracts--
Treponema phenol/water
extracts were separated by HIC. HIC was performed on an FPLC system
(Amersham Pharmacia Biotech, Freiburg, Germany) using an
octyl-Sepharose CL-4B column (Amersham Pharmacia Biotech). An
increasing linear gradient (15-60%) of propanol-1 in ammonium acetate
buffer (pH 4.7) was used as elution buffer with a flow rate of 0.25 ml/min. 500 µl of each fraction was dried and resuspended in 100 µl
of phosphate-buffered saline. For induction of IL-6 in U373MG or
TNF- Expression Plasmids and Transfection of HEK293
Cells--
Expression plasmids for TLR-2 and the dominant-negative
mutants of MyD88 and NIK were generated as described (17). Briefly 3 × 105 HEK293 cells (Tularik, San Francisco, CA)
were cultured in six-well plates with DMEM (Life Technologies Ltd)
supplemented with 10% FCS. HEK293 cells were transfected the following
day by the calcium phosphate precipitation method following the
manufacturer's instructions (CLONTECH, Palo Alto,
CA) with 0.5 µg of ELAM-1 luciferase reporter plasmid, 0.5 µg of
Rous sarcoma virus Preparation and Stimulation of Peritoneal Elicited Macrophages
(PEM)--
Peritoneal macrophages were isolated from C3H/HeJ or
C3H/HeN mice (Charles River, Sulzbach, Germany), by thioglycollate
elicidation. Female 7-week-old mice were injected intraperitoneally
with 1.5 ml of 3% thioglycollate broth (Sifin, Berlin, Germany). After 3 days (C3H/HeN) or after 5 days (C3H/HeJ), mice were sacrificed and
peritoneal macrophages were harvested by injection of 10 ml of ice-cold
Hank's Balanced Salt Solution (Life Technologies) intraperitoneally followed by aspiration. Cells were washed twice with
RPMI 1640, and 1 × 106 cells were plated in 12-well
tissue culture plates in RPMI containing 5% FCS. At the next day,
plates were washed twice with RPMI to remove non-adherent cells, and
remaining cells were stimulated with Treponema glycolipids,
LTAs, or S. minnesota LPS (Sigma) in RPMI containing 2%
non-heat-inactivated FCS for 1 h. Nuclear extracts were prepared
and EMSA was performed as described previously.
Induction of NF-
To examine the role of TLR-2 in responses to Treponema
glycolipids and LTAs from B. subtilis and S. aureus, we used CHO cells transfected with CD14. These cells carry
a frameshift mutation in the TLR-2 gene lacking a functional TLR-2
transcript (58). CHO/CD14 cells respond to exposure of increasing
concentrations of LPS and T. brennaborense by enhanced
translocation of NF- TLR-dependent Induction of Pro-inflammatory Cytokines
by Treponema Glycolipids and LTAs--
Next we addressed the influence
of TLR-2 expression on cytokine induction by Treponema
glycolipids and LTAs by employing the TLR-2-negative human astrocytoma
cell line U373MG (33). IL-6 concentrations in the supernatants were
measured after incubation of U373MG cells with different bacterial
components. LPS and T. brennaborense were effective in
inducing IL-6 production in a dose-dependent manner,
whereas T. maltophilum caused only marginal IL-6 induction.
Both LTAs completely failed to stimulate IL-6 production in U373MG
cells (Fig. 2). In contrast, in the
murine macrophage cell line RAW264.7, both Treponema
glycolipids, LTAs, and LPS induced TNF- Induction of Pro-inflammatory Cytokines in RAW264.7 and U373MG
Cells by Fractions of Treponema Phenol/Water Extracts--
In order to
further analyze the stimulatory capacity of Treponema
glycolipids, they were further fractionated using hydrophobic interaction chromatography. Fractions obtained were tested regarding their ability to induce IL-6 in TLR-2-negative U373MG cells and TNF-
To confirm the different utilization pattern of TLRs by fractions of
T. brennaborense, we investigated the influence of these components on NF- Effect of TLR-2 Overexpression on NF- Involvement of MyD88 and NIK in NF- Induction of NF- Involvement of TLR-4 in RAW264.7 Cell Stimulation by
Treponema Glycolipids and LTAs--
We next examined the
role of TLR-4 in RAW264.7 cell stimulation by using an inhibitory
anti-TLR-4/MD-2 antibody. Therefore, we incubated RAW264.7
macrophages with the TLR-4/MD-2 antibody MTS510 for 1 h
prior to stimulation with glycolipids, LTAs, or S. minnesota
LPS. RAW264.7 cells were exposed to these stimuli for 1 h, and
subsequently nuclear proteins were subjected to EMSA. NF- Induction of NF- NF- The results indicating TLR-2 dependence of LTAs support earlier results
from TLR-2 overexpression experiments in HEK293 cells (38), but are in
contrast to earlier published data indicating TLR-4 dependence of LTAs
(48). In this report commercially available LTAs were used, which were
additionally purified by hydrophobic interaction chromatography, and
which lacked Limulus activity (48). The reason for the
different results may be that, in this report, peritoneal macrophages
from mice were used and it is conceivable that mice TLRs differ in
their binding activity from human or hamster TLRs. Hashimoto et
al. (62) showed previously that only minor macromolecular
glycolipids separated from E. hirae LTA possess cytokine-inducing activity, and that these glycolipids are dependent on
both TLR-2 and -4. Controversial results may be due to different bacterial species used and different LTA extraction methods employed.
Furthermore, our results on TLR utilization obtained with purified
glycolipids from two Treponema strains were confirmed by first experiments with whole treponemes. T. brennaborense
cells displayed TLR-4 dependence, as shown by using an inhibitory
anti-TLR-4 antibody, whereas this effect was less pronounced when using
T. maltophilum bacteria instead. These results obtained with
T. maltophilum cells are in line with published data from
live B. burgdorferi and T. pallidum exhibiting
TLR-2 dependence (34, 63). Similarly, as indicated by our results with
T. brennaborense cells, live Mycobacterium
tuberculosis also activate cells via TLR-2 and TLR-4 (39). In
contrast to M. tuberculosis and T. brennaborense
cells, live Mycobacterium bovis stimulate cells via TLR-2,
but not TLR-4 (39). Our results obtained with whole T. brennaborense bacteria present evidence that lipoproteins, acting
clearly via TLR-2 only (21), are not the only immuno-stimulatory
activity in spirochaetes. Glycolipids acting via TLR-4 as well, as
present in T. brennaborense, may represent an additional
important compound for host-pathogen interaction.
The validity of TLR overexpression experiments in HEK293 cells has been
questioned lately. First results leading to the observation of TLR-2
being a potential LPS receptor now have been contradicted by the fact
that re-purification of the lipopolysaccharide used in these studies
eliminated signaling through TLR-2 in HEK293 cells (17, 64). Several
lines of evidence now clearly support the notion that TLR-4, and not
TLR-2, is the signal transducer of LPS (65, 66). Regardless of these
concerns, the HEK293 cell system still proves to be useful for
functional analysis of microbial TLR ligands. In our studies applying
TLR-2-transfected HEK293 cells, NF- Hydrophobic interaction chromatography has been established for
fractionation LTAs according to their size of the hydrophilic chain
(67). Fractionation of glycolipids from T. brennaborense and
subsequent cell stimulation experiments revealed two different peaks of
activity, one with a clear TLR-2-dependent activity and one
exhibiting TLR-2 independence suggesting TLR-4 utilization.
Chemical analysis of phenol/water extracts from treponemes revealed the
lack of components typically found in LPS such as D-manno-oct-2-ulosonic acid, heptose, and
B activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4.
Fractionation of the T. brennaborense glycolipids by
hydrophobic interaction chromatography and subsequent cell stimulation
experiments revealed two peaks of activity, one exhibiting TLR-2-, and
a second TLR-4-dependence. Furthermore, we show involvement of the
signaling molecules MyD88 and NIK in cell stimulation by LTAs and
glycolipids by dominant negative overexpression experiments. In
summary, the results presented here indicate that TLR-2 is the main
receptor for Treponema glycolipid and LTA-mediated
inflammatory response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)1 into the nucleus
and subsequent activation of responsive genes are important events in
immediate cellular immune response in vertebrates and invertebrates (1). Processes involving activation of NF-
B regulate responses to
stress, inflammation and viral infection, the communication between
cells, embryonic development, and the maintenance of cell type specific
expression of genes. Activation of NF-
B is furthermore required for
transcriptional activation and subsequent release of many mediators
including the pro-inflammatory cytokines interleukin (IL)-1, IL-6, and
tumor necrosis factor (TNF)-
(2).
B activation, i.e. the
IL-1-receptor pathway, encompass a cascade of signal transducing proteins. The adapter protein MyD88 is recruited to the receptor complex upon ligand binding (3, 4). MyD88 recruits the downstream IL-1
receptor-associated kinases IRAK, IRAK-2, and IRAK-M to the receptor
complex (3, 5, 6). The signal is further directed to the adapter
molecule tumor necrosis factor receptor-associated factor-6, the
TGF-
-activated kinase 1, and the NF-
B-inducing kinase (NIK)
(7-10). NIK activates the I
B kinase complex (IKK1/
, IKK2/
,
and NEMO/IKK
), and phosphorylation of I
B triggers degradation and
subsequent nuclear translocation of NF-
B leading to specific gene
activation (9, 11-14).
B (15-22).
The nine currently known human TLRs exhibit homology with the
Drosophila Toll family also comprising at least nine members
(23). The Toll protein was the first member of this protein family
described as a key player in embryonic development and contributes to
defense against fungal infection in the adult fly (24). Another member
of the Toll family in Drosophila, 18-wheeler, has been
suggested to be involved in antibacterial defense (25). Similar to the
IL-1-induced NF-
B induction, TLRs initiated the signaling cascade by
the so-called intracellular cytoplasmic Toll/interleukin-1 receptor
domain TLRs have in common with the IL-1 receptor (15, 26). The
extracellular domains of Toll and TLRs contain leucine-rich repeat
motives (26). TLR-4, most likely in connection with the adapter
molecule MD-2, is the signal transducing receptor for lipopolysaccharide (LPS) from Gram-negative bacteria (19, 27-30), whereas TLR-2 mediates cellular activation by bacterial lipoproteins, whole Gram-positive bacteria, and yeast (31-39). Recently,
peptidoglycan has been shown to be recognized by TLR-2 and -6 (40),
whereas TLR-9 mediates detection of bacterial DNA (41).
B in response to B. subtilis, Streptococcus pyogenes, and S. sanguis LTAs (38). Recently, an
improved isolation procedure employing butanol has been shown to result
in pure and biologically highly active LTAs (49).
in a CD14- and
lipopolysaccharide-binding protein-dependent fashion
(53).
B
activation by these stimuli is induced mainly by TLR-2, however, with
additional utilization of TLR-4 potentially depending on the chemical
composition of the particular microbial component employed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C) were
inoculated in 3 ml of a culture medium (OMIZ-Pat) as described
previously (51). Bacteria were cultured anaerobically (Anaerogen,
Oxoid, Germany) at 37 °C for 3-4 days. The cultures were then
transferred to a larger volume of OMIZ-Pat (20-100 ml) and further
incubated for 1-2 days. Viability of the treponemes and exclusion of
contaminating bacteria were assessed by dark field microscopy (400-fold
magnification, BH2-RFCA microscope, Olympus, Hamburg, Germany).
Sterility controls of the medium were performed by incubating OMIZ-Pat
medium under aerobic and anaerobic conditions at 37 °C for 1 week.
The pH value of the culture medium was measured repeatedly. Cultures
were stopped at pH 6.0 and centrifuged at 12,000 × g
at 4 °C for 20 min. For some experiments, at this point whole
treponeme cells were resuspended in H2O, washed twice, and frozen.
99%.
B
Translocation--
1.6 × 106 RAW264.7 cells/well
were cultured in six-well tissue culture plates overnight in RPMI 1640 (Life Technologies Ltd, Paisley, United Kingdom) containing 10% fetal
calf serum (FCS). After two washing steps with RPMI 1640, cells were
starved for 3 h in the absence of FCS. Stimulation with whole
treponemes, Treponema glycolipids, LTAs from B. subtilis and S. aureus, Escherichia coli
0111:B4 LPS (Sigma), or Streptococcus minnesota LPS (Sigma) for 1 h was performed in the presence of 2% non-heat-inactivated FCS. In certain experiments, RAW264.7 cells were incubated with the
anti-TLR-4-MD-2 antibody MTS510 (30), at concentrations of 5 µg/ml
for 1 h prior to stimulation.
B in the extracts was determined by a standard
electrophoretic mobility shift assay (EMSA). Briefly, 4 µg of the
extracts were incubated with radiolabeled double-stranded oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3', containing the consensus NF-
B DNA site, and electrophoresis was performed on a 4%
polyacrylamide gel.
, 5 × 104 cells/well of RAW264.7 cells were cultured overnight in
96-well tissue culture plates. The cells were stimulated with
Treponema extracts, LTAs, or E. coli 0111:B4 LPS
(Sigma) in the presence of 2% non-heat-inactivated FCS, and
supernatants were harvested after 4 h of incubation. MaxiSorp
ELISA plates (Nunc, Roskilde, Denmark) were coated with 3 µg/ml
anti-mTNF Ab (PharMingen, Heidelberg, Germany) in 100 mM
Na3PO4, pH 6.0. Samples and recombinant mTNF
standard (R&D Systems, Wiesbaden, Germany) were incubated at room
temperature for 3 h, and detection was performed with 500 ng/ml
biotin-conjugated anti mTNF-
Ab (PharMingen), and 1 µg/ml
streptavidin-peroxidase with ortho-phenylene diphosphate
(OPD) as substrate (Sigma).
ELISA.
in RAW264.7 cells, 10 µl of each fraction were used. To
induce NF-
B in CHO/CD14 or RAW264.7 cells, 10 µl of each fraction
40-60 or 61-80 were mixed and cells were incubated with 2 µl of the
pooled fractions.
-galactosidase plasmid to normalize for
transfection efficiency, and 0.2 µg of empty vector or TLR-2
expression vector. In some experiments 0.1 µg of dominant-negative
mutants of MyD88 or NIK were transfected. On the following day, cells
were incubated with Treponema glycolipids or LTAs in the
presence of 2% non-heat-inactivated FCS for 6 h, and luciferase
and
-galactosidase activity was measured by using the Luciferase
Reporter-Gene Assay high sensitivity and the
-Gal Reporter-Gene
Assay from Roche Diagnostics, Mannheim, Germany.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in the Murine Macrophage RAW264.7 and CHO Cell
Line by Treponema Glycolipids and LTAs from B. subtilis and S. aureus--
First we examined the ability of glycolipids from T. brennaborense and T. maltophilum, LTAs from B. subtilis and S. aureus, and E. coli 0111:B4
LPS to induce nuclear translocation of NF-
B in different cell lines.
We exposed the murine macrophage cell line RAW264.7 to increasing
concentrations of different bacterial components for 1 h and
subsequently subjected the nuclear proteins to EMSA.
Treponema glycolipids, LTAs, and LPS caused NF-
B
activation in RAW264.7 cells (Fig. 1).
T. brennaborense exhibited a slightly increased ability to
stimulate these cells as compared with T. maltophilum. A
mock extract including all media and chemicals used during phenol/water
extraction of treponemes failed to exhibit significant stimulation.
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Fig. 1.
Glycolipids and LTAs induce
NF- B translocation in RAW264.7 and CHO/CD14
cells. RAW264.7 and CHO/CD14 cells were stimulated with
concentrations as indicated of glycolipids from T. brennaborense (Tbr) and T. maltophilum
(Tma) and LTAs from B. subtilis (Bs)
and S. aureus (Sa), in comparison to mock extract
or E. coli 0111:B4 LPS in the presence of 2%
non-heat-inactivated FCS. Cells were stimulated for 1 h and
nuclear extracts were prepared as described under "Experimental
Procedures." Extracts were incubated with a radiolabeled DNA probe
containing a NF-
B binding site. NF-
B binding activity was
determined by EMSA. One representative out of three experiments with
similar results is shown (n. d., not determined).
B. In contrast, T. maltophilum and
both LTAs failed to induce NF-
B even at higher concentrations (Fig.
1).
production as expected (data
not shown).
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Fig. 2.
Induction of pro-inflammatory cytokines by
glycolipids and LTAs. U373MG cells were stimulated with increasing
amounts of Treponema glycolipids (Tbr and
Tma), LTAs (S. aureus (Sa) and
B. subtilis (Bs)) or E. coli 0111:B4
LPS in comparison to mock extract. Concentrations of IL-6 in U373MG
supernatants were measured by ELISA. Mean ± S.D. is shown for two
representative experiments in which each stimulation was performed in
three wells.
in RAW264.7 cells (Fig. 3). Fractions
40-60 from T. brennaborense exhibited TLR-2 dependence,
indicated by their ability to stimulate RAW264.7 cells and their
inability to stimulate U373MG cells for cytokine release (Fig. 3A).
However, fractions 61-80 stimulated both cell lines, suggesting the
existence of TLR-2-independent stimulatory molecules present in these
fractions. Fractions 50-80 from T. maltophilum activated
RAW264.7 cells, but completely failed to activate U373MG cells.
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Fig. 3.
Purification of glycolipids from T. brennaborense and T. maltophilum
by octyl-Sepharose column and induction of
TNF- and IL-6 by these fractions.
Treponema phenol/water extracts were fractionated on HIC as described
under "Experimental Procedures." T. brennaborense
(A) and T. maltophilum
(B) fractions were collected and tested for their
ability to induce IL-6 in U373MG cells and TNF-
in RAW264.7 cells.
T. brennaborense fractions 40-60 strongly activated
RAW264.7 cells, but failed to activate U373MG cells (hatched
area).
B activation in CHO/CD14 and RAW264.7 cells. To
this end we stimulated both cell lines with pooled fractions 40-60 and
61-80. In CHO/CD14 cells only fractions 61-80 induced NF-
B
translocation, whereas fractions 40-60 failed to do so (Fig. 4). In RAW264.7 cells both groups of
fractions, 40-60 and 61-80, were similarly able to induce
NF-
B.
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Fig. 4.
Induction of NF- B
translocation in CHO/CD14 and RAW264.7 cells by T. brennaborense fractions. Indicated fractions of
T. brennaborense were mixed and 2 µl were used to
stimulate CHO/CD14 and RAW264.7 cells in the presence of 2%
non-heat-inactivated FCS. After 1 h, nuclear extracts were
prepared and EMSA was performed as described under "Experimental
Procedures."
B Activation by Treponema
Glycolipids and LTAs--
To confirm the finding that glycolipids and
LTAs stimulate cells via TLR-2, we performed overexpression experiments
with the human embryonic kidney cell line HEK293. We transiently
transfected HEK293 cells with a NF-
B-dependent ELAM
promoter luciferase reporter construct. Treponema
glycolipids and LTAs from B. subtilis and S. aureus caused no significant induction of reporter gene activity if a control vector was transfected only (Fig.
5, A and B). After transient transfection of TLR-2, Treponema phenol/water
extracts and both LTAs were able to activate the reporter gene (Fig. 5, A and B).
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Fig. 5.
Effects of TLR-2 overexpression on
NF- B activation induced by
Treponema glycolipids and LTAs. HEK293 cells were
transiently transfected with 0.2 µg of empty control vector or TLR-2
expression plasmid, 0.5 µg of NF-
B-dependent ELAM-1
luciferase reporter plasmid, and 0.5 µg of Rous sarcoma virus
-galactosidase plasmid. Cells were stimulated with the indicated
concentrations of Treponema glycolipids (Tbr and
Tma) (A) and LTAs (B. subtilis
(Bs) and S. aureus (Sa))
(B) in the presence of 2% non-heat-inactivated FCS. After
6 h luciferase activities were obtained by luciferase assay and
normalized with
-galactosidase activity. Data are shown as mean ± S.E. for one representative experiment of three, with transfection
performed in duplicate. Conc., concentration.
B Activation by Treponema
Glycolipids and LTAs--
Following our observation that TLRs are
required for cell stimulation caused by Treponema
glycolipids and LTAs, we investigated the involvement of two downstream
signaling molecules of Toll-like receptors, MyD88 and NIK. Vectors
containing cDNAs for expression of dominant-negative mutants of the
adapter molecule MyD88 or NF-
B-inducing kinase NIK were used in
transient cotransfection assays in HEK293 cells. Overexpression of both
mutants inhibited reporter gene activation by T. maltophilum, T. brennaborense, and LTAs,
suggesting their involvement in TLR signaling pathway leading to
activation of NF-
B (Fig. 6,
A and B).
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[in a new window]
Fig. 6.
Involvement of MyD88 and NIK in
NF- B activation by glycolipids and LTAs.
HEK293 cells were transiently transfected with 0.2 µg of TLR-2
expression plasmid, 0.5 µg of NF-
B-dependent ELAM-1
luciferase reporter plasmid, 0.5 µg of Rous sarcoma virus
-galactosidase plasmid, and 0.2 µg of dominant-negative mutants of
MyD88 or NIK. Cells were stimulated with 100 µg of T. brennaborense and 100 µg of T. maltophilum in
comparison to a mock extract (m) (A) or with 10 µg of LTAs from B. subtilis (Bs) and S. aureus (Sa) in the presence of 2% non-heat-inactivated
FCS (B). After 6 h luciferase activity was monitored as
described under "Experimental Procedures" and normalized with
-galactosidase activity. Activity is shown as mean ± S.E. for
one representative experiment of three, with transfection performed in
duplicate.
B by Treponema Glycolipids and LTAs in
Peritoneal Macrophages Derived from C3H/HeJ and C3H/HeN Mice--
The
LPS hyporesponsive C3H/HeJ mice has been shown to bear a single amino
acid mutation in the cytosolic domain of TLR-4 (19). In order to
analyze a potential TLR-4 utilization in host cell stimulation by
Treponema glycolipids or LTAs, peritoneal macrophages (PEM)
from C3H/HeJ and C3H/HeN mice were stimulated for 1 h, and NF-
B
nuclear translocation was assessed. T. maltophilum
glycolipids led to a comparable induction of NF-
B nuclear
translocation in C3H/HeJ and C3H/HeN PEM (Fig.
7). In contrast, T. brennaborense induced NF-
B activation in C3H/HeJ macrophages
was weaker as compared with C3H/HeN cells, suggesting a
TLR-4-dependent stimulatory activity. LTAs from
S. aureus and B. subtilis exhibited a
strong stimulatory activity in C3H/HeJ PEM. As expected, S. minnesota LPS failed to stimulate PEM derived from C3H/HeJ mice,
but strongly induced NF-
B translocation in C3H/HeN cells.
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[in a new window]
Fig. 7.
NF- B activation in
C3H/HeJ and C3H/HeN macrophages by glycolipids and LTAs.
Peritoneal macrophages from C3H/HeJ mice were stimulated with indicated
concentrations of Treponema glycolipids, 10 µg/ml LTAs,
and 10 ng/ml LPS, and NF-
B activity in the cellular nuclear extracts
was determined by EMSA. In comparison, NF-
B activity in C3H/HeN
macrophages induced by stimulation with glycolipids or LPS is shown
(n. d., not determined). Bs, B. subtilis; Sa, S. aureus.
B
activation caused by T. brennaborense and LPS was inhibited by the TLR-4/MD-2 antibody, indicating TLR-4 dependence (Fig. 8). In contrast, TLR-4/MD-2 antibodies
failed to impair NF-
B translocation in RAW264.7 cells induced by
T. maltophilum and LTAs. To further confirm TLR-4
involvement in signaling by T. brennaborense, the
TLR-2-negative U373MG cell line was stimulated in the presence of an
anti-human TLR-4 antibody (HTA125). As expected, blockade of TLR-4 by
HTA125 led to inhibition of T. brennaborense-induced IL-6
release from U373MG cells (data not shown).
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Fig. 8.
Effects of anti-TLR-4 Ab on
NF- B activation by glycolipids and LTAs.
RAW 264.7 cells were incubated with 5 µg/ml anti-TLR-4-MD-2 Ab
(MTS510) for 1 h prior to stimulation with 1 µg/ml
Treponema glycolipids (Tbr and Tma),
0.1 µg/ml B. subtilis LTA (Bs), S. aureus LTA (Sa), or 0.1 ng/ml S. minnesota
LPS in the presence of 2% non-heat-inactivated FCS. Nuclear extracts
were prepared and incubated with a specific probe containing NF-
B
binding sites, and NF-
B activity was determined by electrophoretic
mobility shift assay. One representative experiment of out of three
with similar results is shown.
B in RAW264.7 Cells by Whole Treponemes--
To
determine the immunostimulatory potential and TLR utilization pattern
of whole treponemes, a first set of experiments with whole bacteria was
performed. We incubated RAW264.7 macrophages with T. brennaborense and T. maltophilum cells, as well as with LPS as control. Cells were pre-incubated with an inhibitory TLR-4/MD-2 antibody (MTS510, kindly provided by Dr. K. Miyake, Saga, Japan) for
1 h, as indicated. After stimulation, the NF-
B translocation was assessed by EMSA. Concentrations of 10 µg/ml T. brennaborense cells induced a pronounced NF-
B translocation in
RAW264.7 cells (Fig. 9). The
anti-TLR-4/MD-2 antibody inhibited the NF-
B induction by T. brennaborense cells and LPS, whereas the effect of the antibody on
NF-
B translocation brought about by T. maltophilum cells
was less pronounced.
View larger version (100K):
[in a new window]
Fig. 9.
Treponeme cells induce
NF- B translocation in RAW264.7
macrophages. RAW264.7 macrophages were pre-incubated with
anti-TLR-4-MD-2 Ab (MTS510) for 1 h, as indicated, and stimulated
with two concentrations of whole T. brennaborense and
T. maltophilum cells, and 1 ng/ml S. minnesota
LPS, in the presence of 2% non-heat-inactivated FCS. After 1 h,
NF-
B translocation was measured by EMSA as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B has been shown to be activated by several microbial
ligands, such as LPS, Treponema pallidum, and
Borrelia burgdorferi lipoproteins and peptidoglycan
(59-61). We demonstrate here that whole treponemes,
Treponema glycolipids, and butanol-extracted LTAs from
S. aureus and B. subtilis are able to activate
NF-
B in murine macrophages indicating their pro-inflammatory
capacity. TLR-2 and -4 are members of the Toll-like receptor family
that have recently been implicated in LTA signaling. Results obtained by employing different TLR-2-negative cell lines, macrophages from
C3H/HeJ mice, and inhibitory anti-TLR-4 antibodies shown here revealed
TLR-2 to be crucial for responses to LTAs and T. maltophilum
glycolipids. In contrast, signaling induced by T. brennaborense glycolipids appears to be dependent on both TLR-2 and -4, confirming earlier results from C3H/HeJ and C3H/HeN mice (53).
B-dependent ELAM
luciferase activity was inducible by Treponema glycolipids
and LTAs. Both LTAs and T. maltophilum activated these cells
in a stronger fashion than T. brennaborense, underlining our
findings with CHO/CD14 and U373MG cells. This assay was furthermore
helpful in indicating the involvement of MyD88 and NIK in signaling by
glycolipids and LTAs leading to activation of NF-
B.
-hydroxylated fatty acids (53). On the other hand, the cell membrane
components isolated displayed LTA-like elements such as sugar, high
phosphate, and alanine similar to that previously identified in
Treponema denticola (68). This similarity was further
supported by isolation and analysis of the dephosphorylated glycosyl
part of the repeating units, which exhibited a low number of large
repeats composing 20-30 sugars in T. maltophilum and a high
number of small repeats containing ~5 sugars in T. brennaborense. Moreover, in T. maltophilum we were able
to identify the lipid anchor composed of two monoacetylated diacylglycerols. These chemical data and the similar biological activities of Treponema phenol/water extracts and LTAs let
us conclude that in T. maltophilum and T. brennaborense LTA-like glycolipids are the major membrane
components responsible for the various biological effects observed in
the extracts (Table I) and probably also
in the whole cells.
Comparison of Treponema glycolipids and LTAs
T. maltophilum glycolipids and both LTAs exhibit no
TLR-4-dependent activity; since both Treponema
glycolipids were extracted in a similar way, a relevant endotoxin
contamination of all bacterial components can be excluded (53). Little
has been known about structural requirements for selective TLR-2 and -4 utilization. TLR-2 is known to be activated by many different ligands
like peptidoglycan, bacterial lipoproteins, whole Gram-positive
bacteria, and yeast (31-39). Here we show that all types of
glycolipids and LTAs employed here are TLR-2 ligands. T. brennaborense exhibited additional TLR-4 dependence, confirming
the previously reported hypothesis of broad ligand specificity for
TLR-2 and a higher selectivity for TLR-4 (34, 35).
Lipoproteins/lipopeptides from B. burgdorferi, T. pallidum, and Mycoplasma fermentans were found to
activate cells via TLRs. All lipoproteins/lipopeptides activated
TLR-2-expressing cells, and acylation of the spirochetal proteins was
critical for their activation of TLR-2. It was thus hypothesized that
amphipathicity may be important for TLR-2 signaling (34). In agreement
with this hypothesis, T. maltophilum and both LTAs
exhibiting TLR-2 dependence revealed higher fatty acid and less
carbohydrate content in our chemical analysis as compared with the
TLR-4 and -2 ligand T. brennaborense (Table I). Furthermore, the glycerophosphate chain from T. maltophilum, B. subtilis, and S. aureus carries alanine residues, which
have been described to lead to charge compensation (43, 44, 53, 69). On
the other hand the more hydrophilic T. brennaborense
fractions, generated in HIC, seem to stimulate cells exclusively via
TLR-2, whereas the more hydrophobic T. brennaborense
fractions seem to utilize TLR-4. Further structural analyses concerning
the biological activity of the different LTAs led us to speculate
whether differences in lipid anchors causing different TLR pattern, or
possibly, a high number of smaller "repeating units" as found in
T. brennaborense result in more "LPS-like" structure
potentially responsible for a TLR-4 utilization. A more detailed
chemical analysis currently performed in our laboratories will enable
us to prove or disprove this interpretation. In summary, our data
provide evidence that TLR-2 is the major receptor for
Treponema glycolipids and LTAs. Additional TLR-4 involvement
may depend on structural elements present in the glycolipids of
T. brennaborense.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Miyake (Saga Medical School, Saga, Japan) for generously providing us with the anti-TLR-4 antibody. Furthermore, we acknowledge Dr. D. Pfeil (Charité) for helpful discussions and Fränzi Creutzburg, Michaela Müller, Nicole Siegemund, Marco Kachler, and Cyndi Hefenbrock (Charité) for outstanding technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Schu 828/1-5 (to R. R. S.), by Bundesministerium für Bildung und Forschung Grants 01 KI 94750 (to R. R. S.) and 01 KI 9318 (to U. B. G.), and by a grant from the Boehringer Ingelheim Fund (to K. S. M.).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: Inst. für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, Dorotheenstr. 96, 10117 Berlin, Germany. Tel.: 49-30-450-524034; Fax: 49-30-450-524900; E-mail: ralf.schumann@charite.de.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M010481200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
EMSA, electrophoretic mobility shift assay;
HIC, hydrophobic interaction chromatography;
I
B, inhibitory
B;
IKK, inhibitory
B kinase;
IL, interleukin;
IRAK, interleukin-1
receptor-associated kinase;
LPS, lipopolysaccharide;
LTA, lipoteichoic
acid;
MyD88, myeloid differentiation protein;
NIK, nuclear factor
B-inducing kinase;
OMIZ-Pat, Treponema culture medium;
PEM, peritoneal macrophage;
TLR, Toll-like receptor;
TNF, tumor
necrosis factor;
Ab, antibody;
CHO, Chinese hamster ovary;
FCS, fetal
calf serum;
ELISA, enzyme-linked immunosorbent assay.
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