From the Institut für Mikrobiologie und
Hygiene, Universitätsklinikum "Charité," Medizinische
Fakultät der Humboldt-Universität zu Berlin,
Dorotheenstrasse 96, D-10117 Berlin, Germany and the ¶ Labor
für Immunchemie, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany
Received for publication, October 2, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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We have shown previously that phenol/water
extracts derived from two novel Treponema species,
Treponema maltophilum, and Treponema brennaborense, resembling lipoteichoic acid (LTA), induce
cytokines in mononuclear cells. This response was lipopolysaccharide
binding-protein (LBP)-dependent and involved Toll-like
receptors (TLRs). Here we show that secretion of tumor necrosis
factor- Spirochetes are involved in a number of chronic inflammatory
diseases, i.e. Treponema pallidum causing
syphilis and Borrelia burgdorferi causing Lyme disease.
These diseases are characterized by certain inflammatory reactions of
the host evoked by the pathogens (1, 2). A possible explanation for
these findings is the presence of cell wall compounds, which, after
being released by the bacteria, induce the secretion of
pro-inflammatory cytokines by host cells, such as macrophages (3). Such
pathways are well established for a range of bacterial compounds,
including lipopolysaccharide (LPS)1 of Gram-negative
bacteria, or LTA and peptidoglycan of Gram-positive bacteria (4-7).
Lipoglycans isolated from spirochetes have been found to be chemically
distinct from LPS (8, 9), and the recent completion of the genome
analysis of T. pallidum revealed the absence of any known
LPS synthesis genes (10). Other investigators implicated outer membrane
lipoproteins of T. pallidum and B. burgdorferi to
be responsible for activation of host cells (11-13). During the last
years, it has been shown by others and ourselves that certain treponeme
species are associated with periodontitis (14-19). This chronic
inflammatory disease is characterized by an inflammatory reaction
followed by extensive loss of tissue. Macrophages are assumed to be
responsible for the inflammatory reactions seen in the host (20), and
cell wall compounds released by spirochetes have been shown to mediate
the production of pro-inflammatory cytokines, i.e. TNF- In previous studies we have investigated the biological characteristics
of phenol/water preparations of the outer membrane of T. maltophilum (TM), a spirochete found in periodontal lesions, and
Treponema brennaborense (TB), a spirochete associated with dermatitis digitalis in cattle, a chronic inflammation of the heels
associated with cachexia (17). We were able to chemically characterize
these preparations by gas liquid chromatography-mass spectroscopy as
lipoglycans due to the presence of a diacylglycerol lipid anchor in TM,
and the presence of repeating carbohydrate units in both strains (22),
resembling LTA of Gram-positive bacteria. These data are in agreement
with a recent study analyzing outer membrane lipoglycans of
Treponema denticola (23). We could show that these
compounds, which are released by bacteria during cell growth, are able
to elicit strong cytokine release by myelo-monocytic cells involving
CD14, LBP, and the Toll-like receptor (TLR) family (22). This pathway
resembles that known for LPS of Gram-negative bacteria (4, 24-26), but
chemical analysis of the treponeme preparations revealed a structure
being clearly distinct from LPS (22).
The aim of this study was to elucidate the signaling pathways leading
to cytokine induction in myelo-monocytic cells caused by treponeme LTA.
It is well established that activation of macrophages caused by LPS is
mediated by LBP, which transfers it to its cellular receptor consisting
of CD14, TLR-4, and the MD-2 molecule (27-29). LPS apparently
initiates signaling via TLR-4 (30-32), while peptidoglycan, as well as
bacterial lipoproteins, and lipoarabinomannan initiate immune responses
via TLR-2 (33-36). It has remained controversial, however, whether LTA
stimulates cell via TLR-2 or -4 (37, 38). Downstream signaling elements
activated by LPS include Ras (39, 40), which, as well as protein kinase
C, leads to activation of the protein kinase Raf1 (39, 41-45), which
in turn activates mitogen-activated protein kinase (MAPK) kinases.
MAPKs p42 and p44 also referred to as "extracellular stress-related
kinase" (ERK)-1/ERK-2 are activated by MAPK/ERK kinase 1 (MEK1)
(46-49). p38, a kinase with high homology to high osmolarity glycerol
response protein 1 of Saccharomyces cerevisiae (50-53), has
been shown to be activated in response to LPS (54), mediated by a MAPK
kinase homologue, MAPK kinase 3 (55). Another family of proteins
closely related to MAPKs are the stress-activated protein kinases
(SAPKs), a member of which is the c-Jun N-terminal kinases (JNKs). They are involved in a range of cellular responses, including UV irradiation (56), as well as LPS activation (57). These three groups of kinases
lead to cytokine production by activating a number of transcription
factors, including c-Jun (activated by JNK), ATF 2 (activated by p38
and JNK), and Elk-1 (activated by p42/44) (58-60).
In this study, we show that treponeme LTA share the ability of parallel
activation of multiple tyrosine-kinase cascades with LPS. Furthermore,
involvement of TLR-2 and -4 in activation of these pathways, employing
the neutralizing anti-murine TLR-4 antibody MTS 510 and the
TLR-2-negative human astrocytoma cell line U373, was investigated.
Treponeme Culture and Processing of Culture
Supernatants--
Frozen stocks of a suspension of TB and TM (300 µl, stored at Extraction of Whole Treponema Cells--
For an extraction of
treponeme whole cells, aqueous suspensions of treponeme cells
were digested with RNase (Sigma, Deisenhofen, Germany), DNase
(Merck, Darmstadt, Germany), and proteinase K (Merck). These
suspensions were dialyzed and further extracted employing a hot
phenol/water extraction method (61). 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 3000 × g for 10 min at 0 °C, and the upper phase was collected.
This procedure was repeated twice; combined phases were dialyzed and lyophilized. A mock extract, including all enzymes and chemicals used,
but no bacteria, was also prepared.
Stimulation of the Murine Macrophage Cell Line RAW 264.7 and the
Human Astrocytoma Cell Line U373--
To assess induction of TNF-
The previously described lack of TLR-2 on U373 cells (34) was confirmed
in our laboratory by reverse transcription-polymerase chain reaction.
For stimulation experiments, U373 cells were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc., Eggenstein, Germany)
containing 10% FCS. For studies on activation of p42/44, 3 × 105 cells/well or 6 × 104 cells/well were
cultured overnight in 6- or 24-well tissue culture plates,
respectively. Prior to stimulation, cells were starved in Dulbecco's
modified Eagle's medium without FCS for 2 h, followed by
stimulation and lysis as described above. For studies on interleukin-6 (IL-6) secretion, 1 × 104 cells/well were cultured
overnight in 96-well tissue culture plates at 37 °C followed by
stimulation. After incubating for 24 h, supernatants were assayed
for IL-6 content as described below.
Detection of Murine TNF- Anti-phosphotyrosine Immunoblotting--
Samples of 50 µg/ml
protein were mixed with sample buffer to obtain a final concentration
of 50 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate
(SDS), 0.1 M dithiothreitol, 10% glycerol, and 0.1%
bromphenol blue, and heated for 5 min at 95 °C. Samples were loaded
onto 12% SDS-polyacrylamide gels, and separated employing a Tris
buffer system. After electrophoresis, gels were immersed in transfer
buffer containing 25 mM Tris-HCl, 0.2 M
glycine, and 20% methanol, and transferred to Hybond-C extra membranes
(Amersham Pharmacia Biotech, Braunschweig, Germany) by semidry blotting (Hölzel GmbH, Dorfen, Germany). For detection of phosphorylated p42/44 and p38 (pp42/44, pp38) membranes were blocked with PBS containing 5% skim milk and 0.1% Tween 20. After washing, blots were
incubated either with rabbit anti-pp38 mAb (Santa Cruz, Heidelberg, Germany) or rabbit anti-pp42/44 mAb (New England Biolabs, Schwalbach, Germany), diluted 1:1000 in PBS, 5% bovine serum albumin (BSA), 0.05%
Tween 20 for 3 h. After washing, membranes were further incubated
with goat anti-rabbit Ab, conjugated with horseradish peroxidase
(Biogenes, Berlin, Germany), diluted 1:5000 in PBS, 5% BSA), 0.05%
Tween 20 for 90 min. After a final washing step, blots were detected
employing the ECL-system (Amersham Pharmacia Biotech) according to the
manufacturer's protocol, and visualized on Hyperfilm ECL-films
(Amersham Pharmacia Biotech). Membranes were stripped by incubation
with 0.1 M glycine, 0.1 M NaCl, pH 2.5, for 30 min at room temperature, and reprobed. For detection of phosphorylated
JNK, membranes were incubated with a mouse mAb directed against
phosphorylated JNK1 and JNK2 (Santa Cruz) diluted 1:100 in
Tris-buffered saline containing 1% BSA, 1% skim milk, and 0.05%
Tween 20, for 3 h. Blocking of membranes and incubation with a
goat anti-mouse IgG Ab conjugated with horseradish peroxidase (Amersham
Pharmacia Biotech) diluted 1:1000, for 90 min, was achieved applying
the same buffer. Blots were visualized as described above employing the
ECL system.
Estimation of p42/44 Activity--
Cell lysates were
investigated for p42/44 activity using the Biotrak system (Amersham
Pharmacia Biotech). In brief, cell lysates were incubated with a
peptide derived from epidermal growth factor receptor highly selective
for p42/44, in the presence of 50 mM [ Expression and Purification of mLBP--
Murine LBP was
expressed in a baculovirus system as described (63). Briefly, the
murine LBP-cDNA was inserted into the baculovirus expression vector
pAcGHLT-B (PharMingen) containing the baculovirus polyhedrin promoter
for high level expression in insect cells, and the glutathione
S-transferase (GST) gene for purification of the resulting
fusion protein. Sf-9 insect cell culture, virus amplification, and
expression of recombinant murine LBP (rmLBP)-GST fusion protein were
performed according to the manufacturer's protocol. Cell lysates were
incubated with GST-Sepharose 4B (Amersham Pharmacia Biotech), and bound
fusion protein was incubated with thrombin (ICN Biochemicals, Eschwege,
Germany) to release mLBP.
Treponema Culture Supernatants Cause Phosphorylation of p42/44,
p38, and JNK1/2--
In previous studies, we have demonstrated that
lipoglycans from TB and TM are released by treponemes during cell
growth. Culture supernatants exhibited a similar stimulation pattern in
comparison to phenol/water extracts of whole cells. To assess
whether the stimulation observed involves phosphorylation of MAPKs, we
incubated RAW 264.7 cells with the supernatants and analyzed the cell
lysates for phosphorylation of these kinases. We observed an
LBP-dependent phosphorylation and activation of p42/44
(Fig. 1, A and B),
as well as phosphorylation of p38 (Fig.
2A). Involvement of the SAPK pathway was analyzed by assessment of JNK1/2 phosphorylation (Fig. 2B). Here also, an LBP-dependent phosphorylation
induced by the culture supernatants was observed. However,
phosphorylation caused by TM-derived culture supernatants was clearly
stronger as compared with TB, although TNF- Treponema LTA Extracted from Whole Cells Reveals a Similar
Phosphorylation Pattern--
Next we performed experiments with LTA
extracted from whole treponeme cells employing a hot phenol/water
method. Cells were stimulated either with treponeme compounds or with
LPS in the presence or absence of recombinant murine LBP for 20 min.
Immunoblotting of the cell lysates revealed a strong phosphorylation of
p42/44 caused by both treponeme LTA and LPS. These reactions were
clearly LBP-dependent (Fig.
3B). The lysates were also
assayed for their state of activation employing the Biotrak p42/44
system, showing a clear correlation between phosphorylation and
activation of the investigated kinases (Fig. 3A). For p38
and JNK, lysates of RAW 264.7 stimulated as described were investigated
for phosphorylation revealing a strong, LBP-dependent
phosphorylation of p38 (Fig. 4A) as well as JNK1 and JNK2
(Fig. 4B) upon stimulation by treponeme extracts. However,
in these experiments, the level of phosphorylation and activation of
kinases failed to differ between the two strains. In all experiments
performed, the "mock" extract did not cause phosphorylation of the
kinases investigated.
Dose Response and Kinetics of Phosphorylation of MAPKs Caused by
Treponeme LTA and LPS in Comparison to TNF- Inhibition of MEK and p38 Leads to Decreased Cytokine
Release--
To determine a crucial involvement of MAPKs p42/44 and
p38 in the events leading to release of TNF- Activation of MAPKs p42/44 by Treponema LTA and LPS in the U373
Astrocytoma Cell Line, and Inhibition of MAPK Activation in RAW 264.7 Cells by the Anti-murine TLR-4 Antibody MTS 510--
TLR-4 has been
shown repeatedly to be associated with LPS signaling, while its role
regarding activation of the MAPK pathway still remains unclear (29). As
we have obtained evidence for a distinct role of TLR-2 and -4 for
cellular activation by the two compounds studied, with TM exhibiting a
TLR-2-dependent NF-
To investigate a potential link between TLR-4 and MAPK activation, we
performed experiments employing the anti-murine TLR-4 antibody MTS 510 known to inhibit LPS effects. Activation of p42/44 in RAW 264.7 caused
by both preparations, as well as by LPS, was clearly decreased in the
presence of the MTS 510 antibody (Fig. 9). The inhibitory capacity of MTS 510 appeared to be more pronounced in regard to TB-induced MAPK activation,
as compared with TM, when low stimulatory concentrations were employed.
However, TNF- In previous studies we have shown that treponeme phenol/water
extracts interact with LBP and CD14, as well as with endotoxin neutralizing protein and polymyxin B, structures commonly viewed as
specific binding partners of LPS (22). Here we demonstrate that
treponeme compounds are able to elicit parallel phosphorylation of the
three major tyrosine kinase cascades, the p42/44, p38, and the SAPK
pathway. Our data therefore indicate biological similarities between
treponeme phenol/water extracts and LPS, although a chemical analysis
of the investigated compounds revealed a structure being clearly
different from that of LPS while resembling LTA of Gram-positive bacteria, with TM containing a diacylglycerol lipid anchor, which is
also proposed for TB. This is in concordance with an analysis of a
lipoglycan derived from T. denticola, another spirochete associated with periodontitis (23).
The stimulating activity of the two treponeme species analyzed was also
found within culture supernatants. We therefore assume that treponemes
release LTA during cell growth provoking signaling events in host cells
involving similar pathways as LPS. T. maltophilum is
found in lesions of patients suffering from periodontitis (17-19), and
compounds released by these bacteria may be the cause for the constant
and severe inflammatory reactions seen in the host. This reaction
pattern also has been proposed as a key pathogenic principle for
Porphyromonas gingivalis, a Gram-negative bacterium containing LPS, which is also associated with periodontitis (67). T. brennaborense has been found in lesions of the heels of
cattle suffering from dermatitis digitalis, a chronic inflammatory
disease of the foot. Although this disease does not affect any other
organs, it coincides with severe cachexia, causing extensive losses in milk production with severe economic consequences worldwide (15). This
cachexia may be caused by increased production of TNF- According to the results presented here, treponeme LTA and LPS differ
in the time course of phosphorylation of MAPKs. This could indicate the
potential activation of other yet undefined pathways, leading to a
prolonged state of activation of kinases. The result of this prolonged
phosphorylation is yet unclear but fails to be associated with an
increase of cytokine induction. Our data provide evidence that
treponeme LTA, although being recognized by the host as indicated by a
substantial initiation of signaling cascades, induces a weak cytokine
production. TNF- Within the last 2 years, the importance of TLRs for host cell
activation by pathogens has become evident. First experiments revealed
a close link between TLR activation and NF- In contrast to these observations, TM revealed a stronger activation of
p42/44 in TLR-2 negative U373 cells as compared with TB, while IL-6
secretion caused by these stimuli resembled NF- As we have shown previously, chemical analysis of both treponeme
compounds revealed differences regarding length and composition of the
carbohydrate chain. Although TB exhibited a large number of small
repeating units of ~5 sugars in size, TM contained fewer repeating
units, each being composed of 20-30 sugars. Additionally, fatty acid
content of TB appeared to be much lower as compared with TM (22).
Although both LTA preparations induced similar signaling events in
mononuclear cells and interact with LPS-binding proteins such as LBP
and CD14, they appear to utilize TLRs in a divergent manner. Still, in
line with our previous study describing the presence of LTA-like
lipoglycan in T. denticola, the general structure of all
treponeme lipoglycans described by us may be similar (23). We assume
that the presence of a diacylglycerol lipid anchor is responsible for
similar biological activity by interaction with LPS-binding structures,
while the length and composition of the carbohydrate chain may affect
the affinity to the different TLRs. However, additional studies have to
be performed to further elucidate this hypothesis.
In summary we have shown that the MAPK and SAPK pathways are induced
not only by LPS but also by structurally distinct bacterial compounds
such as treponeme LTA. Furthermore, we were able to demonstrate a
connection of TLR-4 and activation of MAPKs p42/p44. Finally, we
obtained evidence for a dissociation of the MAPK and the NF- induced by Treponema culture supernatants and
extracted LTA was paralleled by an LBP-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) p42 and
p44, and p38, as well as the stress-activated protein kinases c-Jun
N-terminal kinases 1 and 2. Phosphorylation of p42/44 correlated with
an increase of activity, and tumor necrosis factor-
levels were
significantly reduced by addition of inhibitors of p42/44 and p38, PD
98059 and SB 203580, respectively. Treponeme LTA differed from
bacterial lipopolysaccharide regarding time course of p42/44 phosphorylation, exhibiting a prolonged activation of MAPKs.
Furthermore, MAPK activation and cytokine induction failed to be
strictly correlated. Involvement of TLR-4 for phosphorylation of p42/44
was shown employing the neutralizing anti-murine TLR-4 antibody MTS
510. In TLR-2-negative U373 cells, the compounds studied differed
regarding MAPK activation with T. maltophilum leading to a
stronger activation. In summary, the data presented here show that
treponeme LTA are able to activate the MAPK and stress-activated
protein kinase pathway involving LBP and TLR-4.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(12, 21).
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 (19) and cultured under anaerobic
conditions (Anaerogen, Oxoid, Germany) at 37 °C for 3-4 days. The
cultures were then transferred to 20-100 ml of OMIZ-Pat and incubated
for another 1-2 days. For phenol/water extraction of whole cells, these cultures were transferred to 500 ml of OMIZ-Pat. Viability of the
treponemes and the exclusion of contaminating bacteria were assessed by
dark field microscopy (400-fold magnification; BH2-RFCA microscope,
Olympus, Hamburg, Germany). Sterility controls of the medium
preparation 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, and the cultures were stopped
at pH 6.0. The protein concentration of the supernatants was determined
employing the Bio-Rad protein assay (Bio-Rad, Munich, Germany).
Concentrations ranged from 1.2 to 1.5 µg/ml (TB), and from 0.4 to 0.6 µg/ml (TM), respectively. Cultures were centrifuged at 12,000 × g for 20 min at 4 °C and passed through 0.2-µm sterile
filters (Schleicher & Schuell, Dassel, Germany). For some studies,
culture supernatants were heat-inactivated at 100 °C for 20 min and
passed again through 0.2-µm sterile filters. OMIZ-Pat medium, treated
similarly, served as control.
,
5 × 104 cells/well of RAW 264.7 cells were
cultured overnight in 96-well tissue culture plates using RPMI 1640 containing 10% FCS. After two washing steps with RPMI 1640, stimulation was performed in the absence or presence of 1 µg/ml
recombinant murine LBP in a total volume of 100 µl. Supernatants were
harvested after 4 h of incubation, and cells were stained with
trypan blue. For some experiments, cells were incubated with the MEK
inhibitor PD 98059 (Calbiochem, Schwalbach, Germany), or the p38
inhibitor SB 203580 (Alexis, Läufingen, Switzerland) at 37 °C
for 1 h prior to stimulation. In other experiments, as indicated,
the inhibitory monoclonal anti-TLR-4 antibody MTS510 (62), kindly
provided by Dr. Miyake (Saga, Japan), was added 30 min prior to
stimulation. To investigate phosphorylation and activity of tyrosine
kinases, 1.6 × 106 cells/well were cultured in
six-well tissue culture plates over night in RPMI 1640 containing 10%
FCS. After two washing steps with RPMI 1640, cells were starved for
3 h in the absence of serum. Stimulation was done in the absence
or presence of 1 µg/ml rmLBP. After 20 min, supernatants were removed
and cells were washed three times with ice-cold PBS containing 1 mM Na3VO4, followed by addition of
150 µl of lysis buffer, containing 1% Triton X-100 (Roth, Karlsruhe,
Germany), 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 2 mM EGTA, 1 mM
phenylmethanesulfonyl fluoride, 0.2 mM leupeptin, and 0.15 unit/ml aprotinin. After 15 min of incubation at 4 °C, cells were
scraped off the plates and centrifuged for 30 min at 12,000 × g at 4 °C. The postmitochondrial supernatant was
recovered, protein content was determined employing the Bio-Rad protein
assay, and the remaining lysates were stored at
80 °C. For some
experiments cells were stimulated for a longer period, as indicated.
and Human Interleukin-6
(IL-6)--
For detection of murine TNF-
, MaxiSorp enzyme-linked
immunosorbent assay plates 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 employing a
biotin-conjugated anti mTNF-
Ab (PharMingen), and
streptavidin-peroxidase with ortho-phenylene diphosphate as
substrate. The detection limit was ~15 pg/ml. For quantification of
IL-6 plates were coated with anti-human IL-6 antibody (R & D Systems).
Samples and recombinant human IL-6 (R & D Systems) were detected
employing a biotin-labeled monoclonal anti-human IL-6 antibody (R & D Systems). The detection limit was 16 pg/ml. Shown are
mean values ± S.D.
-33P]ATP (3, 7 × 103 Bq/test) for
30 min at 36 °C. After separating the peptide from unincorporated
activity, incorporated ATP was measured in a scintillation counter.
Shown are mean values ± S.D.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
induction was similar.
Activity of p42/44 was related to the state of phosphorylation as
revealed by the Biotrak p42/44 activity assay (Fig. 1A).
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Fig. 1.
Phosphorylation and activation of p42/44 by
treponeme culture supernatants. RAW 264.7 cells were stimulated
with 10% treponeme culture supernatants or 10 ng/ml LPS
(Escherichia coli 0111:B4) for 20 min in the presence or
absence of rmLBP. Cells were lysed, and 5 µg of the lysates were
assayed for p42/44 activity employing a Biotrak kinase activity assay
(A). Counts per minute (cpm) on the y axis
represent p42/44 activity. This assay was performed in triplicate. 50 µg of protein were further analyzed by immunoblotting employing an
anti-phospho-p42/44 Ab (B). Blots were stripped and detected
with an antibody directed against unphosphorylated p42 and p44 as a
loading control. Shown is one representative experiment of four with
similar results.
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Fig. 2.
Phosphorylation of p38 and JNK1/2 by
treponeme culture supernatants. RAW 264.7 cells were stimulated
with 10% treponeme culture supernatants, 10% culture medium alone, or
10 ng/ml LPS (E. coli 0111:B4) for 20 min with or without
rmLBP as indicated. Cell lysates (50 µg for p38, 100 µg for JNK1/2)
were blotted, and detection of phosphorylated p38 (A) and
JNK1/2 (B) was performed employing antibodies against their
phosphorylated isoforms as described under "Experimental
Procedures." Antibodies against unphosphorylated p38 and JNK1/2
served as loading controls. Shown is one representative of two
experiments.
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Fig. 3.
Treponema LTA derived from whole
cells induces phosphorylation and activation of MAPKs p42/44. RAW
264.7 cells were stimulated with LTA derived from T. brennaborense or T. maltophilum (1 µg/ml), with a
mock extract, or with LPS (E. coli 0111:B4, 10 ng/ml) for 20 min. rmLBP was added at a concentration of 1 µg/ml as indicated.
Phosphorylation of p42/44 (B) was assessed via
immunoblotting of 50 µg of protein derived from cell lysates. Shown
is one representative experiments out of four. Activation of p42/44 was
assessed using 5 µg of protein derived from the same lysates
(A). The assay was performed in triplicate. Shown is one
representative of four experiments.
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Fig. 4.
Phosphorylation of p38 and JNK1/2 by
treponeme LTA. RAW 264.7 cells were stimulated with treponeme LTA
(1 µg/ml), mock extract, or LPS (E. coli 0111:B4, 10 ng/ml) for 20 min with or without rmLBP. Cell lysates (50 µg for p38,
100 µg for JNK1/2) were blotted, and detection of phosphorylated p38
(A) and JNK1/2 (B) was performed employing
antibodies against their phosphorylated isoforms. Antibodies against
unphosphorylated p38 and JNK1/2 served as a loading control. Shown is
one representative of two experiments.
Release--
In
previous studies treponeme phenol/water extracts were found to be less
potent regarding cytokine induction as compared with LPS. To obtain
TNF-
concentrations comparable to those elicited by LPS, the
treponeme preparations were concentrated ~1000-fold. To compare the
dose dependence of MAPK activation with that found in TNF-
release,
cells were stimulated with treponeme LTA or LPS for assessment of
p42/44 phosphorylation, or TNF-
release, respectively. TNF-
release induced by LPS was significantly stronger as compared with
treponeme LTA-induced activation (Fig.
5A). For phosphorylation
studies, cells were stimulated with LPS or treponeme LTA, and
activation of p42/44 was assessed. LPS exhibited a stronger activation
of kinases; however, at the concentrations used, all stimuli exhibited
a comparable state of activation (Fig. 5, B and
C). It has been shown that LPS-induced activation of MAPKs reaches its maximum after 15 min (64). To compare kinetics of MAPK
activation induced by treponemes and LPS, we stimulated RAW 265.7 for
different time periods ranging from 10 to 60 min. In these experiments
LPS-induced phosphorylation and activation of MAPKs reached a maximum
at 20 min (Fig. 6B). In
contrast, treponeme LTA exhibited a different time pattern of
phosphorylation and activation of p42/44; for TB activation was
observed after 20 min, but further increased up to 60 min after
stimulation (Fig. 6C). TM exhibited an activation of p42/44
with a maximum at 40 min (Fig. 6D).
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Fig. 5.
Dose-dependent phosphorylation of
p42/44 and TNF- release caused by treponeme
LTA and LPS. RAW 264.7 cells were stimulated with increasing
concentrations of LTA derived from T. brennaborense,
T. maltophilum, mock extract, or 100 ng/ml LPS (E. coli 0111:B4) in the presence of rmLBP (1 µg/ml). Cell lysates
(5 µg of protein) were assayed for activation of p42/44
(A). This assay was performed in duplicate. 50 µg of
lysates were examined for phosphorylation of MAPKs (B);
shown is one representative of two experiments. In parallel, RAW 264.7 cells were stimulated with the same compounds for 4 h and TNF-
content of the supernatants was assessed as described under
"Experimental Procedures" (C). Shown is one
representative of three experiments.
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Fig. 6.
Time course of phosphorylation of p42/44
induced by treponeme LTA in comparison to LPS. RAW 264.7 cells
were stimulated with a mock extract (A), LPS (E. coli 0111:B4, 100 ng/ml, B), or with LTA derived from
T. brennaborense (10 µg/ml, C) or T. maltophilum (10 µg/ml, D) for 1, 10, 20, 40, and 60 min in the presence of rmLBP (1 µg/ml). Cells were lysed, and 50 µg
of protein were examined for phosphorylation of p42/44 by
immunoblotting. Activation of MAPKs was assessed employing the Biotrak
activity assay. Shown is one representative of two experiments. The
activity assay was performed in duplicate.
, RAW 264.7 were
incubated with inhibitors of MEK1 and p38 prior to stimulation, and
TNF-
-concentrations were measured after 4 h. MEK1 has been
shown to activate MAPK p42/44, and inhibition of this kinase leads to
suppression of MAPKs activity (65, 66). Cytokine induction caused by
both treponeme preparations and LPS was strongly reduced in the
presence of both inhibitors (Fig. 7).
When both inhibitors were used simultaneously, TNF-
levels further
decreased. Higher concentrations did not further influence cytokine
induction (data not shown).
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[in a new window]
Fig. 7.
Reduction of TNF-
release by inhibitors of p42/44 and p38. RAW 264.7 cells
were incubated with the MEK inhibitor PD 98059 (50 µM) or
the p38 inhibitor SB 203580 (10 µM) for 1 h at
37 °C prior to stimulation. Cells were stimulated with treponeme LTA
(10 µg/ml) or LPS (E. coli 0111:B4, 10 ng/ml), and
supernatants were harvested after 4 h followed by detection of
mTNF-
. Shown is one representative of three experiments;
measurements were performed in quadruplicate.
B translocation and NO induction
(21), we used two in vitro systems to investigate the role
of TLRs in treponeme LTA-induced MAPK activation. The TLR-2-negative
U373 astrocytoma cell line was stimulated with both treponeme
preparations and LPS, and MAPK activation was assessed (Fig.
8). In contrast to previous observations, TM caused a clear MAPK activation while TB failed to do so. IL-6 production induced by TM, however, appeared to be
TLR-2-dependent as it was less pronounced as compared with
TB (data not shown).
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[in a new window]
Fig. 8.
Activation of MAPKs p42/44 in the human
astrocytoma cell line U373. U373 cells were incubated with 10 µg/ml Treponema LTA, mock extract, or 100 ng/ml LPS,
respectively, for 20 min at 37 °C followed by lysis. 10 µg of
lysates were assayed for p42/44 activity employing the Biotrak kinase
activity assay. Experiments were performed in triplicate; shown are
combined results of two separate experiments.
induction in RAW 264.7 caused by the same stimuli was
not reduced in the presence of MTS 510 (data not shown).
View larger version (26K):
[in a new window]
Fig. 9.
Decrease of activity of MAPKs p42/44 in the
presence of a blocking TLR-4 antibody. RAW 264.7 cells were
incubated with the blocking anti-murine TLR-4 antibody MTS 510 for 30 min at 37 °C, followed by stimulation with Treponema LTA,
mock extract, or LPS (E. coli 0111:B4) for 20 min in the
presence of rmLBP (1 µg/ml). Cell lysates were harvested and
investigated regarding activation of p42/44. Experiments were performed
in triplicate. Shown is one representative of two experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in the host,
elicited by LTA via signaling pathways proposed here. The findings
observed here may also apply for other diseases caused by spirochetes,
such as syphilis (caused by T. pallidum, closely related to
the strains investigated here (Ref. 19)), or Lyme disease.
induction was particularly weak when T. maltophilum-derived preparations were employed; however, LTA of
both strains induced signaling events in a comparable manner.
B translocation in
different cell systems leading to a rapid induction of pro-inflammatory cytokines (68, 69). In contrast, few data exist establishing a clear
link between TLRs and the MAPK/SAPK pathways with one study describing
divergent pathways for SAPKs (70). Here we show for the first time that
inhibiting ligand binding to TLR-4 reduces activation of MAPK p42/44.
This is evidence for a connection of TLR-4 and MAPKs and may,
furthermore, indicate a link between the well established TLR-NF-
B
cascade and the MAPKs. The inhibition experiments presented here
confirm our previous observations of a divergent use of TLRs by the two
different compounds investigated. Although TB in experiments employing
macrophages from TLR-4-defective C3H/HeJ mice appeared to stimulate
cells via TLR-4, TM apparently preferentially utilizes TLR-2, as
indicated by the lack of NF-
B translocation in TLR-2-defective
Chinese hamster ovary cells (22). The inhibition by the anti-TLR-4
antibody observed here was more pronounced for TB as compared with TM
when using low concentrations, further supporting a preferential
utilization of TLR-4 by TB. This effect, however, in our hands could
not be demonstrated for cytokine induction, which may be explained by
the fact that cytokine release had to be assessed at a later time point.
B translocation with
TB being more active as TM. Taken together with our observations that
treponeme LTA, although showing a strong activation of MAPKs in RAW
264.7, only induced a weak TNF-
release, these data could indicate a
dissociation between the NF-
B pathway with subsequent cytokine
release, and the MAPK pathway. On the other hand, our results obtained
with the MEK and p38 inhibitors clearly support the notion that MAPK
activation leads to cytokine release. Thus, although both the MAPK and
NF-
B pathway lead to cytokine induction by bacterial compounds, they
may not be linked in a synergistic manner.
B
pathway in myelo-monocytic cells. The compounds investigated by us
appear to be valuable tools for investigating signaling pathways
involving TLRs, as here structurally related compounds apparently
utilize TLRs in a different manner.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the excellent technical assistance of Nicole Siegemund, Fränzi Creutzburg, Michaela Müller, Marco Kachler, and Cyndi Hefenbrock.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Schu 828/1-5 (to R. R. S.), and by Bundesministerium für Bildung und Forschung Grants 01 KI 94750 (to R. R. S.) and 01/KI 9318 (to U. B. G.).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 the Boehringer Ingelheim Foundation.
To whom correspondence and reprint requests should be
addressed. Tel.: 49-30-2093-4747; Fax: 49-30-2093-4704; E-mail:
ralf.schumann@ charite.de.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M008954200
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ABBREVIATIONS |
---|
The abbreviations used are:
LPS, lipopolysaccharide;
ERK, extracellular stress-related kinase;
IL-6, interleukin-6;
JNK, c-Jun N-terminal kinase;
LBP, lipopolysaccharide-binding protein;
LTA, lipoteichoic acid;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein
kinase/extracellular stress-related kinase kinase;
NF-B, nuclear
factor
B;
OMIZ-Pat, treponeme culture medium;
rmLBP, recombinant
murine LBP;
SAPK, stress-activated protein kinase;
TB, Treponema
brennaborense;
TLR, Toll-like receptor;
TM, Treponema
maltophilum;
TNF, tumor necrosis factor;
Ab, antibody;
mAb, monoclonal antibody;
GST, glutathione S-transferase;
FCS, fetal calf serum;
DTT, dithiothreitol;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin.
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