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INTRODUCTION |
Invasive bacterial infections in mammals may lead to severe
systemic complications such as septic shock, once bacteria overcome the
first line of defense, which is mediated by the innate immune system
(1, 2). During the last decade, the relative number of Gram-positive
bacteria responsible for bloodstream infections has steadily increased,
and recent epidemiological studies revealed that Gram-positive
microorganisms cause the majority of systemic infections in the United
States and Europe (3, 4). Staphylococcus aureus, followed by
Enterococcus spp. in the United States and Streptococcus pneumoniae in Europe, is the organism most
frequently isolated during invasive infections, playing an important
role in hospital infections (3, 4). According to the Centers for
Disease Control, S. pneumoniae (also called pneumococcus) is
the major cause of community-acquired pneumonia, meningitis, and otitis
media in the United States. Furthermore, invasive infections caused by
pneumococci are responsible for about one million deaths in children in
developing countries yearly, comparable with Plasmodium spp.
causing malaria, with respect to the number of incidents (5). During
recent years, spreading of multiresistant staphylococci (i.e. methicillin-resistant S. aureus, MRSA, Ref.
6) and pneumococci (drug-resistant S. pneumoniae, DRSP), has
raised major problems concerning therapy for these infections,
especially in intensive care units in the United States and South
America (7).
For Gram-negative bacteria the pathogenesis of septic shock has been
elucidated with the isolation of its major outer membrane component
lipopolysaccharide (LPS)1
(8-10). LPS, an amphiphilic molecule or glycolipid, is commonly released by bacteria during growth and cell death (11). In the course
of systemic infections caused by Gram-negative bacteria, aggregates of
LPS as well as intact bacterial cells are rapidly opsonized by
LPS-binding protein (LBP), a serum protein synthesized in the liver
(12). LBP effectively catalyzes the transfer of LPS to membrane-bound
and soluble forms of CD14 (mCD14 or sCD14, Refs. 13-15). GPI-anchored
mCD14 is predominantly expressed by cells of the monocytic lineage (16)
and profoundly sensitizes myeloid cells to minute amounts of LPS.
However, it has been predicted to require a co-receptor for the
initiation of cellular signaling caused by the lack of a transmembrane
domain (17, 18). Toll-like receptor (TLR)-4 is a member of a family of
receptors displaying homology in their cytoplasmic domains to the
interleukin-1 receptor (19) and playing an important role in innate
immunity by recognizing molecular patterns of a wide range of bacteria
and protozoan species and molecules related to viral infections (20,
21). TLR-4 has been identified as a specific receptor for LPS leading
to the induction and release of pro-inflammatory cytokines by monocytes and macrophages upon LPS stimulation (22). To induce cytokine release
by monocytes in response to extracellular LPS via TLR-4, however, an
accessory host molecule termed MD-2 is required (23), which binds with
high affinity to the ectodomain of TLR-4 and affects subcellular
distribution of TLR-4 (24).
In contrast, detailed information on the molecular patterns of
Gram-positive bacteria interacting with the innate immune system is
still lacking. In several previous studies two molecules of the cell
wall, peptidoglycan (PG) and lipoteichoic acid (LTA) have been found to
mediate inflammatory responses in the host. PG forms the murein layer
of the bacterial cell wall and has been repeatedly described to
activate immune cells (25-27). LTA represents a class of amphiphilic
molecules anchored to the outer face of the cytoplasmic membrane in
Gram-positive bacteria and is commonly released during cell growth,
especially under antibiotic therapy (28-30). It causes cytokine
induction in mononuclear phagocytes (31, 32), and a synergism with PG
has been described resulting in higher cytokine levels (33, 34). After
the identification of TLRs, a number of controversial reports on the
involvement of TLR-2, TLR-4, and MD-2 in LTA-induced cell activation
have been published (24, 35-37). However, although LTA is meanwhile regarded as an important mediator of inflammation (38), the recruitment
of TLRs is still unclear, because in most of these studies commercial
LTA preparations were used (24, 31, 32, 36), which not only display a
high degree of compositional heterogeneity but are contaminated by
significant amounts of LPS (39, 40). Recently, we have described a
novel purification protocol for LTA from S. aureus based on
a butanol extraction procedure (41). LTA from S. aureus
extracted by this protocol lacks LPS contamination, and being of high
purity was found to strongly induce cytokines in a human whole blood
assay. These results were confirmed by largely analogous data obtained
with chemically synthesized LTA (42).
All LTA described to date exhibit a common molecular architecture
consisting of a diacylglycerol-containing glycolipid anchor and a
covalently coupled polymeric backbone structure (43, 44). However, LTA
from different Gram-positive species have been found to differ in the
chemical composition of the so-called "repeating units" of the
polymeric backbone (44). In S. aureus, the repeating units contain D-alanine and
-D-N-acetylglucosamine linked to a central
linear 1-3-linked polyglycerophosphate chain (45). This
structural motif has also been shown to be valid in LTA from Enterococcus spp., Bacillus subtilis, and
some streptococci (45). In contrast, the polymeric chain of LTA from
S. pneumoniae exhibits a strikingly different polymeric
structure, consisting of tetrasaccharide repeating units that contain
phosphorylcholine and are linked to each other by ribitol phosphate
(46).
Recently it was shown that heat-killed whole cell preparations derived
from S. aureus and S. pneumoniae induce
activation of transcription factor NF-
B in transfected CHO cells in
a TLR-2 and CD14-dependent fashion (47). This is consistent
with the observation that TLR-2-deficient mice are highly susceptible
to infection by S. aureus, compared with the wild type-like
immune response of TLR-4-deficient mice (37). These mice were
furthermore highly susceptible to pneumococcal meningitis (48).
However, the particular TLR signaling pathway activated by LTA from
S. aureus has remained controversial, and no information has
been available as yet on the potential contributions of LBP, CD14, TLR-2 or TLR-4, and MD-2 in the interaction of LTA from S. pneumoniae with the innate immune system.
The aim of this study was to analyze the role of LBP and CD14 in
cytokine induction caused by LTA of S. pneumoniae and
S. aureus. Of further interest was the potential involvement
of TLR-2, TLR-4, and MD-2 in signal transduction by LTA. Our data show
that LBP and CD14 are involved in TLR-2-dependent
initiation of immune responses to both staphylococcal and
pneumococcal LTA.
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EXPERIMENTAL PROCEDURES |
Purification of LTA from S. pneumoniae and S. aureus--
LTA
of S. aureus DSM 20233 (SLTA) was prepared by butanol
extraction followed by purification using HIC chromatography as described previously (41). S. pneumoniae strain R6
(49), kindly provided by E. Tuomanen, St. Jude's Children's
Research Hospital, Memphis, TN, was cultured in CSL medium to late log
phase and harvested by centrifugation at 3000 × g.
Integrity of bacteria and potential contaminations by Gram-negative
bacterial species were checked by Gram staining and microscopy.
Pneumococcal LTA (PLTA) was extracted from defrozen bacterial cells
employing a chloroform/methanol protocol as described earlier (46), and then extracts were purified by HIC chromatography as described for the
LTA of S. aureus (41).
SDS Gel Electrophoresis, Western Blotting, and Staining
Procedures--
Polyacrylamide stacking gels (5%) and separating gels
(16%) were cast without SDS. LPS and LTA preparations were mixed with 4× sample buffer and loaded onto the gel, and electrophoresis was
performed according to Laemmli (68). Gels were stained using the
BioRad Silver Stain Plus kit (BioRad, Munich, Germany) according to the
manufacturers protocol, except that gels were oxidized in 0.7%
periodic acid prior to staining (50). Colloidal gold staining was
performed employing the colloidal gold staining kit (BioRad, Munich,
Germany) following the manufacturer's protocol. For
anti-phosphorylcholine Western blotting, gels were immersed in transfer
buffer containing 25 mmol/liter Tris-HCl, 200 mmol/liter glycine, 20%
methanol, and transferred to Hybond-C extra membranes (Amersham
Biosciences) by semidry blotting (Hölzel GmbH, Dorfen, Germany).
Membranes were blocked in phosphate-buffered saline (PBS) containing
2% bovine serum albumin (Roth, Braunschweig, Germany) for 2 h at
room temperature and washed three times. TEPC-15 (Sigma), a
phosphorylcholine-specific IgA-antibody from murine ascites (51), was
diluted 1:250 in PBS/2% bovine serum albumin and incubated with the
membrane at 4 °C overnight. After washing, a rabbit anti-murine IgA
antibody (Sigma), diluted 1:2000 in PBS/2% bovine serum albumin, was
added and incubated for 1 h at room temperature. After a final
washing step, bands were visualized by the ECL system (Amersham
Biosciences) using Hyperfilm ECL films (Amersham Biosciences) as
recommended by the manufacturer's protocol.
PhastGelTM Native and SDS Electrophoresis--
To
analyze binding of PLTA or SLTA to soluble CD14 (sCD14) and to
investigate effects of LBP on complex formation, an automated form of
non-denaturing gel electrophoresis was performed using the
PhastsystemTM apparatus (Amersham Biosciences). Highly
purified LPS from Escherichia coli 515 was analyzed as a
control. Briefly, LTA or LPS preparations at concentrations of 1.25 or
0.0625 µg/µl, respectively, were incubated in Dulbecco's
phosphate-buffered saline (D-PBS without magnesium
or calcium; Invitrogen) in the absence or presence of 0.25 µg/µl
recombinant human sCD14 (Biometec, Greifswald, Germany) at 37 °C for
10 min. For some experiments recombinant human LBP (Xoma Corp.,
Berkeley, CA) was added at a concentration of 0.025 µg/µl. For
analysis of interaction of LBP with ligands, a set of samples was
analyzed containing LBP at a final concentration of 0.25 µg/µl.
After incubation at 37 °C samples were placed on ice and 4× native
sample buffer (pH 7.6) was added. Native PhastGelTM
electrophoresis was performed at 4 °C by automated application of 1 µl of each sample to PhastGelTM Homogeneous-20 gels
equipped with PhastGelTM native buffer strips.
Electrophoretic separation was done at a constant voltage of 400 V. The
integrity of sCD14 and LBP proteins during the incubation procedure was
verified by adjusting native samples to denaturing conditions with the
addition of SDS to a final concentration of 2%, heating at 95 °C
for 5 min, and PhastGelTM SDS electrophoresis. Following
electrophoresis, automated silver staining of native and SDS gels was
performed according to the manufacturer's protocol.
Isolation and Stimulation of Human Peripheral Blood
Monocytes--
Blood from healthy donors was drawn with heparin (50 units/ml) and diluted 1:2 in RPMI 1640 (Invitrogen). 30 ml were layered on 15-ml Pancoll (PAN Biotech, Aidenbach, Germany) and centrifuged at
600 × g at 21 °C for 15 min. The interphase was
washed two times in RPMI 1640 and centrifuged at 600 × g at 21 °C for 5 min. Thrombocytes were separated by
another centrifugation step at 100 × g at 21 °C for
15 min. Remaining cells were diluted in RPMI 1640 containing 2% human
albumin (Immuno, Heidelberg, Germany) and incubated in 96-well tissue
culture plates for 2 h, followed by three washing steps with RPMI
1640 in order to remove non-adherent cells. Stimulation experiments
were performed in a 100-µl volume in the presence of 2.5% human
serum (Sigma) or recombinant human LBP (Xoma Corp., Berkeley, CA). The
LBP preparation was found to be devoid of any endotoxin contamination
employing a kinetic chromogenic limulus amoebocyte lysate (LAL) test
(Bio Whittaker, Verviers, Belgium). For some experiments, cells were
incubated with anti-CD14 antibody My4 (Coulter, Hamburg, Germany) prior to stimulation at 36 °C for 30 min. Stimulation was performed with
PLTA and SLTA. S-Form LPS derived from Salmonella
minnesota (Sigma) employed by us in earlier studies and shown to
be devoid of any lipoprotein contamination (52) served as a control.
For some experiments, cells were preincubated with muramyldipeptide (MDP, Bachem, King of Prussia, kindly provided by T. Hartung, Konstanz). After 4 h, supernatants were subjected to
ELISA.
Detection of Human Tumor Necrosis Factor-
(TNF-
)--
Nunc
Maxi Sorp ELISA plates were coated with 5 µg/ml rabbit anti-hTNF-
Ab diluted in 100 mM NaHCO3, pH 8.3 at 4 °C
overnight. After blocking with PBS containing 0.05% Tween 20 (Sigma)
and 10% fetal calf serum (FCS, Invitrogen) for 2 h at room
temperature, samples and recombinant TNF-
(R&D, Wiesbaden, Germany)
were added and incubated at 4 °C overnight. After washing,
biotinylated anti-hTNF-
antibody (BD Pharmingen, Hamburg, Germany)
at 5 µg/ml was added and incubated at room temperature for 1 h,
followed by incubation with streptavidin-peroxidase (1 µg/ml, Sigma)
for 30 min. Detection of bound TNF-
was carried out with
ortho-phenylen-diphosphate (OPD, Sigma,) followed by measurement at 490 nm in an ELISA reader (Tecan, Crailsheim, Germany).
Culture and Transfection of HEK293 Cells--
Wild-type HEK293
cells and cells stably transfected with human CD14 were cultured in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10%
FCS, transfectants were additionally supplemented with 400 µg/ml G418
(Invitrogen). Prior to transfection, cells were transferred to 12-well
tissue culture plates at 3.5 × 104 cells per well and
cultured overnight. After washing, cells were transfected with plasmids
encoding for hTLR-2 (0.002 µg), hTLR-4 (0.002 µg), hMD-2
(0.002-0.05 µg)
-galactosidase (0.1 µg), and the ELAM NF-
B
luciferase reporter plasmid (0.25 µg) employing 4 µl/well
LipofectAMINE® transfection reagent (Invitrogen). All
plasmids were kindly provided by C. J. Kirschning, Technical
University Munich, Munich, Germany (69) except the hMD-2 plasmid, which
was provided by K. Miyake, University of Tokyo, Tokyo, Japan. After
4 h, medium was changed, and cells were cultured in DMEM/10% FCS
overnight. Stimulation was performed in DMEM without FCS for
16 h. Cells were lysed, and
-galactosidase and luciferase
activity were measured using a kit based on chemiluminescence (Roche
Molecular Biochemicals).
Cultivation and Transfection of Chinese Hamster Ovary (CHO)
Cells--
Wild-type CHO cells stably transfected with human CD14 (53)
were cultured in Ham's nutrient medium F12 (PAA, Linz, Austria), and
transfectants were additionally supplemented with 400 µg/ml G418
(Invitrogen). For stimulation experiments, cells were cultured at a
density of 5 × 104 cells/well in 12-well tissue
culture plates without G418 overnight. Cells were washed with Ham's
medium without FCS and transfection with expression plasmids encoding
for hTLR-2 (0.01 µg), hCD14 (0.01 µg),
-galactosidase (0.1 µg), and the ELAM NF-
B luciferase reporter plasmid (0.25 µg) for
16 h was performed employing 0.8 µl/well FuGENE®
(Roche Molecular Biochemicals). Cells were washed and stimulated with
LPS and LTA preparations in 1 ml of Ham's medium for 24 h followed by lysis and measurement of
-galactosidase and luciferase activity. For some experiments, CHO cells expressing a mutant MD-2
defective in LPS-induced signaling (54) were cultivated in Ham's
medium containing 100 µg/ml Hygromycin (Invitrogen).
Statistical Analysis--
Enhancing effects of LBP, CD14, TLR-2,
and TLR-4 as well as inhibitory effects of anti-CD14 Ab My4 were
statistically evaluated employing the Student's t test.
Throughout the figures, p values of <0.05 are indicated by
one asterisk, p values of <0.01 by double asterisks.
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RESULTS |
Analysis of Pneumococcal Lipoteichoic Acid--
Isolation
procedures for pneumococcal LTA (PLTA) differ from any other known
protocols for purification of LTA (46, 55). To assess the purity and
the structural integrity of the PLTA preparation extracted with
chloroform/methanol and purified on octyl Sepharose, we performed
periodic acid-sensitized silver stain analysis and
anti-phosphorylcholine Western blotting (Fig. 1). LTA derived from staphylococci
displays a faint staining pattern when treated with silver staining
reagents. Because of the higher carbohydrate content of PLTA, it
exhibited a stronger staining pattern displaying two distinct major
bands of ~31 and 36 kDa in size. These failed to stain with gold
colloid, indicating the absence of protein contamination (Fig. 1).
Employing a phosphorylcholine-specific Western blot, the presence of
phosphorylcholine could be demonstrated for the 31 and 36 kDa-size
bands of the PLTA preparation as well as for a series of minor bands
distributed in the 17-40 kDa range (Fig. 1C). These bands
most likely represent LTA molecules of different size present at lower
quantities within the preparation. In fact, all bands detected by
periodic acid-sensitized silver staining were also stained by the
phosphorylcholine-specific immunodetection method.

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Fig. 1.
Purity and structural integrity of LTA
derived from S. pneumoniae. LTA derived from S. pneumoniae (PLTA) and S. aureus (SLTA), as well as LPS
of S. minnesota wild-type (10 µg per lane) were separated
on 16% polyacrylamide gels by electrophoresis according to Laemmli.
Gels were subjected to periodate-sensitized silver staining
(A), colloidal gold staining (B), or blotted and
detected employing the phosphorylcholine-specific antibody TEPC-15
(C).
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Induction of TNF-
in Human Monocytes by LTA--
First, we
tested the potential pro-inflammatory activation of human mononuclear
phagocytes by LTA employing stimulation experiments with isolated
peripheral blood monocytes (PBMCs) in the absence of serum. Both PLTA
and SLTA induced TNF-
in a dose-dependent manner without
substantial differences in the activation profiles (Fig.
2A). As compared with LPS,
cytokine levels induced by LTA were lower, and under serum-free
conditions, LTA had to be used at concentrations of up to 3 orders of
magnitude greater compared with LPS to elicit comparable levels of
TNF-
. Since synergistic effects of LTA and peptidoglycan partial
structures in the activation of murine macrophages and human monocytic
cells have been reported in prior studies (33, 34), we tested whether
preincubation of human monocytes with muramyldipeptide (MDP) affected
cytokine release by LTA. Both preparations (SLTA and PLTA) led to
profoundly higher levels of TNF-
release when monocytes were
preincubated with MDP at 100 ng/ml compared with LTA alone (Fig. 2,
B and C).

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Fig. 2.
Induction of TNF- by
LTA and LPS in human monocytes in the absence of serum:
synergisms of PLTA and SLTA with muramyldipeptide. A,
human monocytes isolated from peripheral blood were stimulated with
increasing concentrations of PLTA and SLTA, as well as LPS of S. minnesota in the absence of human serum and TNF- was estimated
after 4 h by ELISA. B and C, human monocytes
were incubated with increasing concentrations of SLTA and PLTA
(C) with or without preincubation with MDP (100 ng/ml) for
30 min at 36 °C prior to stimulation. TNF- was estimated after
4 h by ELISA. Experiments were performed in duplicates. Shown are
representatives of three experiments with similar results.
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Effect of Serum, Recombinant Human LBP, and CD14--
The presence
of 2.5% human serum markedly enhanced the stimulatory potency of both
SLTA and PLTA (Fig. 3). Since serum
enhanced the stimulatory activity of LTA toward monocytes in a
comparable manner, as was well documented for LPS (12, 13), we
hypothesized that LBP may be one of the major serum components
mediating this effect for LTA. When PBMCs were stimulated with PLTA and
SLTA in the presence of LBP in concentrations ranging from 0.1 to 10 µg/ml, significant enhancing effects on cytokine induction by both
LTAs were detected (Fig. 3C). This effect varied according to the bacterial source of LTA and was dose-dependent for
LBP. When PLTA concentrations of 0.4 µg/ml were employed LBP
significantly increased the stimulation of cytokine release. For SLTA,
at both concentrations tested pronounced enhancing effects of LBP on
TNF-
release were observed in comparison to the non LBP-treated
control. Notably, for PLTA at 0.4 µg/ml and SLTA at 0.4 µg/ml and 2 µg/ml a maximal enhancing effect was obtained at an LBP concentration of 1 µg/ml whereas 10 µg/ml led to an enhanced, but significantly lower degree of monocyte activation. A similar activity profile was
obtained for the enhancing effects of LBP in LPS-induced TNF-
release that was found to be maximal at the lowest LBP concentration tested and less pronounced at higher LBP concentrations of 1 and 10 µg/ml, respectively (Fig. 3C). Next, the potential
involvement of CD14 in LTA-induced monocyte activation was tested
employing the blocking anti-CD14 monoclonal antibody My4. Cytokine
levels elicited in response to SLTA in the presence of LBP were
significantly reduced by addition of My4 (Fig.
4A), and for PLTA the same
characteristics were observed (Fig. 4B). LPS, in contrast to
LTA, stimulated cells independently of CD14 when higher concentrations
were tested (Fig. 4C). Comparable results were obtained when
blocking effects were investigated in the presence of serum instead of
recombinant LBP (Fig. 4D).

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Fig. 3.
Effect of serum and recombinant human LBP on
LTA and LPS-mediated cytokine induction in human monocytes. Human
peripheral blood monocytes were stimulated with increasing
concentrations of SLTA (A), PLTA (B), or LPS of
S. minnesota wild-type (C) in the absence or
presence of serum, and TNF- levels were estimated by ELISA after
4 h of incubation. D, human monocytes were stimulated
with PLTA, SLTA, and LPS in the presence of increasing concentrations
of recombinant human LBP. Experiments were performed in duplicates
(A-C) or quadruplicates (D). Shown are
representatives of three experiments with similar results.
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Fig. 4.
Effect of anti-CD14 antibody My4 on cytokine
induction in human monocytes by LTA and LPS. Human monocytes were
stimulated with increasing concentrations of PLTA (A), SLTA
(B), or LPS (C) in the presence of recombinant
human LBP with or without preincubation with the blocking anti-CD14
antibody My4 (1 µg/ml) for 30 min at 36 °C prior to addition of
stimuli. D, monocytes were stimulated with PLTA, SLTA, and
LPS in the presence of 5% human serum with or without preincubation
with My4. Experiments were performed in duplicates (A-C) or
triplicates (D). Shown are representatives of three
experiments with similar results.
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Gel-shift Analysis on the Interaction of LTA with sCD14 and
LBP--
As the data obtained from monocyte activation experiments
indicated a modulation of activity of LTA from S. aureus and
S. pneumoniae by LBP and CD14, we tested the potential
direct interaction of LTA with LBP and CD14 (Fig.
5). In contrast to sCD14 and LBP, LTA
preparations were not detected directly by automated silver staining of
the native gels, whereas LPS was stained by this protocol in the
absence of sCD14 or LBP as a band in the upper region of the separating
gel. In the presence of PLTA, a shift of sCD14 toward higher mobility
was observed, combined with the additional detection of a slower
migrating band (Fig. 5A). The sCD14 band of increased
electrophoretic mobility was clearly more pronounced when LBP, at a
substoichiometric concentration of 25 ng/ml (0.42 pmol/ml) compared
with sCD14 (5 pmol/ml), was added, indicating a catalytic transfer of
PLTA to sCD14 by LBP (lane 4). Comparable effects were
observed with SLTA (Fig. 5B). Similarly, in the case for
PLTA, addition of LBP slightly enhanced the formation of the upper
retarded band. For SLTA the intensity of this second band was
significantly reduced compared with the sample containing sCD14 and LTA
without LBP. LBP alone at a concentration of 250 ng/ml (4.2 pmol/ml)
also displayed a shift toward higher mobility (Fig. 5, A and
B, lane 7) after incubation with PLTA and SLTA. Here, LBP-mediated catalytic transfer of LPS toward sCD14 was also
observed (Fig. 5C), consistent with previous results from others (56).

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Fig. 5.
Gelshift assay for interaction of LTA with
CD14 and LBP and enhancement of LTA-CD14 complexation by LBP binding of
PLTA (A) and SLTA (B) to recombinant
human sCD14 or LBP. The modulating effects of LBP on complexation
of LTA and sCD14 were analyzed by native PhastGelTM
electrophoresis in comparison to LPS from E. coli F515
(C). Following incubation at 37 °C for 10 min, samples
were subjected to automated native electrophoresis on 20%
PhastGelsTM and subsequent silver staining employing the
PhastSystemTM apparatus. The position of uncomplexed sCD14
is indicated by an arrow. In panels A and
B the positions of the corresponding sCD14-LTA complexes of
increased (1) and retarded (2) electrophoretic mobility as well as of
the LBP complexes of PLTA and SLTA (3) are additionally marked by
stars. As loading control, the native samples were
subsequently adjusted to a final concentration of 2% (w/v) SDS, and
additionally analyzed by PhastGelTM SDS electrophoresis and
silver staining (lower panels).
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LTA Stimulates HEK293 Cells and CHO Cells via TLR-2--
In
previous studies we demonstrated the involvement of TLR-2 in signaling
events caused by butanol-extracted SLTA (57). Here, we employed HEK293
cells stably transfected with human CD14 and transiently transfected
with human MD-2 and combinations of MD-2 with TLR-2 or TLR-4. In this
system, PLTA and SLTA acted as ligands for TLR-2, because only
transfection with TLR-2 led to enhanced activation of the cells upon
stimulation with either of the LTA preparations (Fig.
6) while transfection of TLR-4 and MD-2
displayed no effect. Furthermore, CHO cells, which were shown previously to lack functional TLR-2 because of a point mutation, expressing TLR-4 and MD-2 (58) were employed. CHO cells were stably
transfected with human CD14 followed by transient transfection with
human MD-2 alone or with combinations of TLR-2 and MD-2. Results
confirmed the observations obtained with transfected HEK293 cells
indicating that a functional TLR-2 is required for cellular response to
LTA while TLR-4 and MD-2 are not sufficient (Fig. 6, D and
E). Overexpression of CD14 in addition to TLR-2 in wild-type CHO cells significantly enhanced the TLR-2-dependent
activation of NF-
B by PLTA and SLTA, confirming the involvement of
CD14 in LTA recognition (Fig. 6F).

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Fig. 6.
Requirement of TLR-2 and CD14 in the
activation of HEK293 and CHO cells by LTA. HEK293/CD14 cells were
transiently transfected with plasmids encoding for -galactosidase
and a luciferase reporter construct (ELAM). In addition, plasmids
encoding for MD-2 (A), MD-2 and TLR-2 (B), or
MD-2 and TLR-4 (C) were transfected employing
LipofectAMINE®, followed by stimulation with PLTA, SLTA,
or LPS for 16 h. Cells were lysed, and chemiluminiscence
indicating -galactosidase and luciferase activity was estimated.
Activation is indicated using arbitrary units
( -galactosidase/luciferase × 10). CHO/CD14 cells were
transiently transfected with plasmids encoding for -galactosidase
and a luciferase reporter construct (ELAM). In addition, the cells were
transfected with MD-2 (D) or MD-2 and TLR-2 (E)
employing FuGENE®, followed by stimulation with PLTA, SLTA (500 ng/ml
each), or LPS (S. minnesota, 100 ng/ml) for 16 h.
Activation of cells was estimated as described above. F, CHO
wild-type cells were transiently transfected with a plasmid encoding
-galactosidase, a luciferase reporter construct (ELAM), human MD-2,
and TLR-2, with or without a plasmid encoding human CD14. Cells were
stimulated, and activation was estimated as indicated above.
Experiments were performed in triplicates; shown are representatives
from three experiments with similar results.
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LTA-mediated Activation of HEK293 Cells Does Not Require
MD-2--
Two recent studies reported a crucial role for MD-2 in
LTA-mediated cell activation, one describing an
MD-2-dependent cellular activation by LTA via TLR-2 and
TLR-4 (35), and one via TLR-4 only (24). In both studies LTA from
S. aureus and other Gram-positive bacteria was prepared by a
hot phenol/water extraction procedure. In previous experiments we
failed to detect any interaction of our LTA preparations with TLR-4;
therefore, we tried to determine whether MD-2 had any effect on the
translocation of NF-
B in HEK293/CD14 cells transiently transfected
with human TLR-2 or human TLR-4. Additional expression of human MD-2 in
TLR-2-transfected HEK293/CD14 cells did not affect unstimulated cells
and had no significant effect on PLTA and SLTA NF-
B activation (Fig.
7). We also tested lower LTA
concentrations, but MD-2 effects were never observed (data not shown).
In the LPS control, signaling was significantly enhanced by
cotransfection of MD-2 in TLR-4-transfected HEK293/CD14 cells (Fig.
7B); however, increasing amounts of MD-2 led to a decline of
LPS-dependent stimulation. To further verify the lack of
MD-2 involvement in TLR-2-mediated cell activation by LTA, we used a
CHO cell line defective in MD-2 expression (54). These cells were
completely unresponsive to LTA (Fig. 7C). Transient transfection with human TLR-2 restored LTA-mediated activation, but
additional transfection of human MD-2 failed to increase the NF-
B
translocation caused by LTA in line with the results obtained for
HEK293/CD14 cells.

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Fig. 7.
Effect of MD-2 on activation of HEK293 cells
by LTA. HEK293/CD14 cells were transiently transfected with
plasmids encoding for -galactosidase, a luciferase reporter
construct (ELAM) and TLR-2 (A) or TLR-4 (B).
Additionally, cells were transfected with increasing amounts of a
plasmid encoding for MD-2. Stimulation and estimation of activation
were performed as indicated above. Experiments were performed in
triplicates; shown are representatives from three experiments.
C, CHO cells expressing a non-LPS responsive mutant of
Chinese hamster MD-2 were transfected with human TLR-2 alone or with
human TLR-2 and MD-2 together with CD14, -galactosidase and ELAM for
24 h employing FuGENE®. Cells were stimulated with PLTA or SLTA
for 16 h, followed by measurement of luciferase activity. Data are
presented as -fold increase of stimulation as compared with the
controls. Experiments were performed in triplicates; shown is one
representative of two experiments.
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DISCUSSION |
Gram-positive bacteria have become the leading cause of hospital
infections and play a major role in community acquired infectious diseases. Unfortunately, the molecular patterns causing inflammatory responses induced by these organisms are still not defined. Regarding innate immune responses to Gram-positive bacteria, biological activities of several components derived from these microbes have been
reported (31, 32, 35, 36). However, no model has been widely accepted,
and systemic complications resulting from infections with Gram-positive
bacteria, e.g. sepsis and septic shock have often been
attributed to LPS, as a consequence of shock-induced translocation of
Gram-negative bacteria (59). The major problem in investigating
Gram-positive bacteria and their immunostimulatory compounds is the
lack of homogenous material of high purity, devoid of LPS
contamination. For the preparation of LTA a variety of protocols exist.
The majority of previous studies was performed employing commercial
preparations of LTA (31, 32, 36), which recently have been found to be
of high heterogeneity and to contain substantial amounts of LPS (39,
40). Therefore, data providing reliable information on biological
activities of LTA are rather scarce.
The preparations employed in this study were purified by a novel
butanol extraction protocol (S. aureus) and a
chloroform/methanol procedure (S. pneumoniae), respectively.
As compared with the widely used phenol/water procedure, both protocols
work at lower temperatures, which for S. aureus LTA has been
shown to be an important factor for retaining biological activity,
while yielding a material of high purity (41). The purity of PLTA was
verified by parallel application of periodate-sensitized silver
staining, colloidal gold staining, and phosphorylcholine-specific
immunodetection, revealing the absence of contaminating protein as well
as the presence of bands detectable with phosphorylcholine-specific
antibody. As has been shown for SLTA (41), PLTA represents a highly
purified preparation.
Both LTA preparations induced cytokines in human PBMCs. In the presence
of serum cytokine induction by LTA was greatly enhanced. As compared
with LPS, LTA preparations by concentration were less active; however,
the tested concentrations of LTA (1 µg~107
colony-forming units) as well as of LPS (20 ng~107
colony-forming units) are comparable in order of magnitude of bacterial
cell equivalents (60). In earlier studies synergistic effects of PG
partial structures with LTA have been reported employing crude
preparations as well as purified or synthetic MDP (33, 34, 61). Here,
we provide evidence that the highly purified PLTA and SLTA preparations
act in synergy with MDP to induce high cytokine levels. Recent evidence
suggests that in the case of MDP and LPS this synergy involves the
induction of an increased cytoplasmic accumulation of TNF
mRNA
by MDP and a subsequent TLR-4-dependent triggering of
translation and cytokine release by LPS as the second stimulus
(61).
Previous studies indicated an interaction of LBP with cell wall
structures of apathogenic B. subtilis; however, in this
study phenol/water-extracts were employed (62). Our previous results revealed that murine LBP interacted with butanol-extracted LTA of
S. aureus and B. subtilis by use of a microplate
binding assay (63), while no modulating effects of murine LBP toward
the stimulatory effects of LTA on human monocytes were observed. This
discrepancy can be attributed to the different origin of LBP used in
the studies. In addition, residual LBP from serum may have masked the
effects of added LBP on LTA-induced activation in our previous study
that involved a different cell isolation protocol. However, here we observed an involvement of both LBP and CD14 in cellular activation by
LTA from S. aureus and S. pneumoniae. These data
are in line with our studies on outer membrane glycolipids of
Treponema spp. sharing structural similarities with LTA (64,
65). Because all these molecules differ greatly with respect to the
structure of their polymeric backbone, it is tempting to speculate that interaction with CD14 and LBP, in analogy to LPS, occurs via the more
conserved lipid anchor. Furthermore, we observed a marked reduction of
LTA-mediated cytokine release by LBP at high concentrations. This
observation parallels data on the effects of high levels of LBP, as
present during the acute phase, in murine in vivo models of
LPS-induced lethality and Gram-negative bacteremia as well as in human
monocyte activation, published by us previously (66, 67). These results
suggest that increased serum levels of LBP may protect the host from
cytokine-mediated systemic complications during sepsis induced by
Gram-positive bacteria.
Both LTA preparations were shown to increase electrophoretic mobility
of sCD14 as compared with the uncomplexed protein, indicating an
interaction between these structures. The addition of LTA to LBP also
induced an increased electrophoretic mobility of the LBP band in our
assay, indicating the formation of stable LBP-LTA complexes. In analogy
to sCD14-LPS complexation described earlier (56), formation of the
sCD14 complex with PLTA or SLTA was also found to be enhanced in the
presence of LBP, indicating a transfer of LTA to sCD14 by LBP. However,
the coincubation of either PLTA or SLTA with sCD14 additionally induced
the formation of a second band displaying a retarded electrophoretic
mobility in the native PAGE system. Currently, we can only speculate
that the latter band may represent a sCD14-LTA complex containing
either one or both of the ligands in higher stoichiometries. The data
obtained with native PAGE analysis support our observations on the
enhancing functions of LBP and CD14 in the LTA-induced inflammatory
activation of host cells and suggest that the lipid transfer protein
functions of LBP may also include the opsonization of LTA micelles and
the catalysis of LTA binding to CD14.
In previous studies we were able to show that LTA purified from
S. aureus and B. subtilis activated cellular
responses via TLR-2 (57). In this study, we extended this observation
to LTA from pneumococci employing two different cell lines. We
performed a series of transfection experiments to analyze the potential contribution of MD-2 to LTA-induced cellular signaling and found that
NF-
B translocation caused by LTA was not dependent on MD-2. Since
co-transfection of TLR-4 with MD-2 also failed to render cells
responsive to LTA, TLR-4 and MD-2, in contrast to their essential roles
in signal induction by LPS (23, 24), can be ruled out as being involved
in LTA-mediated signaling. This conclusion was complemented and
confirmed by the use of MD-2-negative CHO-cells. Because of these
observations, we are further able to rule out that the preparations
tested were contaminated with LPS.
Employing purified preparations of two major structural forms of LTA
representing clinically highly relevant Gram-positive bacteria we
provide further evidence that LTA is a potent mediator of innate immune
responses. The LTA recognition pathway of the host in some aspects
parallels the well documented system of the host's LPS recognition.
According to several lines of evidence we conclude that activation of
cellular responses by LTA is mediated by TLR-2 and is enhanced by LBP
and CD14, but is clearly independent of TLR-4 and MD-2. These data
should help in understanding the molecular mechanisms involved in the
pathogenesis of infections caused by Gram-positive bacteria and their
systemic complications, and may potentially help in developing novel
intervention strategies.