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Lipoteichoic Acid (LTA) of S. pneumoniae and S. aureus Activates Immune Cells via

Toll-like Receptor (TLR)-2, Lipopolysaccharide (LPS) Binding Protein (LBP) and CD14

while TLR-4 and MD-2 are not Involved

Nicolas W. J. Schröder*, Siegfried Morath°, Christian Alexander†, Lutz Hamann*, Thomas

Hartung°, Ulrich Zähringer†, Ulf B. Göbel*, Joerg R. Weber§, and Ralf R. Schumann*

*Institut für Mikrobiologie und Hygiene,

Universitätsklinikum "Charité"

Medizinische Fakultät der Humboldt-Universität zu Berlin

Dorotheenstr. 96, D-10117 Berlin, Germany

° Biochemische Pharmakologie

Universität Konstanz

Box M 668, D-78457 Konstanz, Germany

† Abteilung Immunchemie und Medizinische Mikrobiologie

Forschungszentrum Borstel, Zentrum für Medizin und Biowissenschaften

Parkallee 22, D-23845 Borstel, Germany

§ Neurologische Klinik,

Universitätsklinikum "Charité"

Medizinische Fakultät der Humboldt-Universität zu Berlin

Schumannstr. 20/21, D-10098 Berlin, Germany

Running Title: Innate Immune Responses to S. pneumoniae and S. aureus

Key Words: lipoteichoic acid, cytokines, toll-like receptors, lipopolysaccharide-

binding protein, CD14

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 19, 2003 as Manuscript M212829200 by guest on January 24, 2020

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Summary

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Summary

Lipoteichoic acid (LTA) derived from Streptococcus pneumoniae, purified employing a

chloroform/methanol protocol and from Staphylococcus aureus, prepared by the recently

described butanol-extraction procedure, were investigated regarding their interaction with

lipopolysaccharide (LPS)-binding protein (LBP), CD14, Toll-like receptors (TLRs)-2 and -4,

and MD-2. LTA from both organisms induced cytokine synthesis in human mononuclear

phagocytes. Activation was LBP- and CD14-dependent, and formation of complexes of LTA

with LBP and soluble CD14 (sCD14) as well as catalytic transfer of LTA to CD14 by LBP

was verified by PhastGel native gel electrophoresis. Human embryonic kidney (HEK)

293/CD14 cells and Chinese hamster ovary (CHO) cells were responsive to LTA only after

transfection with TLR-2. Additional transfection with MD-2 did not affect stimulation of

these cells by LTA. Our data suggest that innate immunity recognition of LTA via LBP,

CD14 and TLR-2 represents an important mechanism in the pathogenesis of systemic

complications in the course of infectious diseases brought about by the clinically most

important Gram-positive pathogens, while an involvement of TLR-4 and MD-2 is ruled out.

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Introduction

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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, mediated by the innate immune

system (1,2). During the last decade, the relative number of Gram-positive bacteria

responsible for blood stream 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 US 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 US. Furthermore,

invasive infections caused by pneumococci are responsible for about 1 million deaths in

children in developing countries yearly, thus being 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 Staphylococcus aureus, MRSA (6)) and

pneumococci (drug resistant Streptococcus pneumoniae, DRSP), has raised major problems

concerning therapy of these infections, especially in intensive care units in the US and South

America (7).

For Gram-negative bacteria the pathogenesis of septic shock has been elucidated since the

isolation of its major outer membrane component, lipopolysaccharide (LPS) (8-10). LPS, an

amphiphilic molecule or glycolipid, is commonly released by the 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

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Introduction

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protein (LBP), a serum protein synthesized in the liver (12). LBP effectively catalyses the

transfer of LPS to membrane-bound and soluble forms of CD14 (mCD14 or sCD14, (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 due to the lack of a

transmembrane domain (17,18). Toll like receptor (TLR)-4 is a member of a family of

receptors displaying homologies 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, protozoan species and also molecules related to viral infections

(20,21). TLR-4 has been identified to act 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 represent 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

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Introduction

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the identification of TLRs, a number of controversial reports on the involvement of TLR-2, –4

and MD-2 in LTA-induced cell activation have been published (24,35-37). However,

although LTA are meanwhile regarded as important mediators of inflammation (38), the

recruitment of TLRs in these mechanisms is still unclear, as in most of these studies

commercial LTA-preparations were used (24,31,32,36) that 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 lacking

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 diacyl-

glycerol-containing glycolipid anchor and a covalently coupled polymeric backbone structure

(43,44). However, LTA from different Gram-positive species have been found to vary in

chemical composition of the so-called “repeating units” of the polymeric backbone (44). In S.

aureus, the repeating units contain D-alanine and α-D-N-acetylglucosamine linked to a

central linear 1-3-linked poly-glycerophosphate chain (45), and this structural principle has

also been shown to be valid in LTA from Enterococcus spp., B. 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 which contain

phosphorylcholine and are linked to each other by ribitolphosphate (46).

Recently it was shown that heat-killed whole cell preparations derived from S. aureus and S.

pneumoniae induce activation of transcription factor NF-κB in transfected CHO cells in a

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Introduction

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TLR-2 and CD14-dependent fashion (47). This is concordant with the observation that TLR-

2-deficient mice are highly susceptible to infection by S. aureus as compared to a wildtype-

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 so far 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, -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., while TLR-4 and MD-2 are not involved.

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Experimental Procedures

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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 by 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, USA, was cultured in CSL medium to late log phase and harvested by centrifugation at

3000 x 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), additionally, extracts were purified by HIC-chromatography as

described for LTA of S. aureus (41).

SDS Gel Electrophoresis, Western Blotting and Staining Procedures

Polyacrylamide stacking gels (5%) and separating gels (16%) were cast without sodium

dodecyl-sulfate (SDS). LPS and LTA preparations were mixed with 4 x sample buffer, loaded

on the gel, and electrophoresis was performed according to Laemmli. 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 BioRad colloidal gold staining kit (BioRad,

Munich, Germany) following the manufacturers protocol. For anti-phosphorylcholine

Western Blotting, gels were immersed in transfer buffer containing 25 mmol/L Tris-HCl, 200

mmol/L Glycine and 20 % methanol, and transferred to Hybond-C extra membranes

(Amersham, Braunschweig, Germany) by semi-dry blotting (Hölzel GmbH, Dorfen,

Germany). Membranes were blocked in phosphate buffered saline (PBS) containing 2%

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bovine serum albumin (BSA, Roth, Braunschweig, Germany) for 2 h at room temperature and

washed three times. TEPC-15 (Sigma, Deisenhofen, Germany), a phosphorylcholine-specific

IgA-antibody from murine ascites (51), was diluted 1:250 in PBS/2% BSA and incubated

with the membrane at 4 °C over night. After washing, a rabbit anti murine IgA antibody

(Sigma, Deisenhofen, Germany), diluted 1:2000 in PBS/2% BSA, was added and incubated

for 1 h at room temperature. After a final washing step, bands were visualized by the ECL-

system (Amersham, Braunschweig, Germany) as recommended the manufacturers protocol

using Hyperfilm ECL-films (Amersham, Braunschweig, Germany).

PhastGel™ 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 Phastsystem™ apparatus (Amersham Pharmacia Biotech). Highly

purified LPS from E. coli 515 was analyzed as a control. Briefly, LTA or LPS preparations at

concentrations of 1.25 µg/µl or 0.0625 µg/µl, respectively, were incubated in Dulbecco´s

phosphate-buffered saline (D-PBS w/o magnesium or calcium; Life Technologies/GIBCO,

BRL, Germany) 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

4x native sample buffer (pH 7,6) was added. Native PhastGel™ electrophoresis was

performed at 4 °C by automated application of 1 µl of each sample to PhastGel™

Homogeneous-20 gels equipped with PhastGel™ native buffer strips. Electrophoretic

separation was done at a constant voltage of 400 V. The integrity of sCD14 and LBP proteins

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during the incubation procedure was verified by adjusting native samples to denaturing

conditions by addition of SDS to a final concentration of 2 %, heating at 95 °C for 5 min. and

PhastGel™ 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 U/ml) and diluted 1:2 in RPMI 1640

(Gibco, Eggenstein, Germany). 30 ml were layered on 15 ml Pancoll (PAN Biotech,

Aidenbach, Germany) and spun at 600 x g at 21 °C for 15 min. The interphase was washed

two times in RPMI and spun at 600 x g at 21 °C for 5 min. Thrombocytes were separated by

another centrifugation step at 100 x g at 21 °C for 15 min. Remaining cells were diluted in

RPMI 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 in order to

remove non-adherent cells. Stimulation experiments were performed in 100 µl volume, for

some studies in the presence of 2.5 % human serum (Sigma, Deisenhofen, Germany) 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 S. minnesota (Sigma, Deisenhofen, Germany) 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, USA, kindly provided by T. Hartung, Konstanz,). After 4 h, supernatants were

subjected to ELISA.

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Experimental Procedures

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Detection of Human Tumor Necrosis Factor-α (TNF-α)

Nunc Maxi Sorp ELISA plates were coated with 5 µg/ml rabbit anti-hTNF-α Ab diluted in

100 mM NaHCO3, pH 8,3 at 4 °C over night. After blocking with PBS containing 0,05 %

Tween 20 (Sigma, Deisenhofen, Germany) and 10 % fetal calf serum (FCS, Gibco,

Eggenstein, Germany) for 2 h at room temperature, samples and recombinant TNF-α (R&D,

Wiesbaden, Germany) were added and incubated at 4 °C over night. After washing,

biotinylated anti-hTNF-α antibody (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, Deisenhofen, Germany) for 30 min. Detection of bound TNF-α

was carried out with ortho-phenylen-diphosphate (OPD, Sigma, Deisenhofen, Germany)

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 medium (DMEM, Gibco, Eggenstein, Germany) containing 10 %

FCS, transfectants were additionally supplemented with 400 µg/ml G418 (Gibco, Eggenstein,

Germany). Prior to transfection, cells were transferred to 12 well tissue culture plates at 3,5 x

104 cells per well and cultured over night. 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) ß-galactosidase

(0,1 µg) and the ELAM NF-κB luciferase reporter plasmid (0,25 µg) employing 4 µl/well

Lipofectamine® transfection reagent (Invitrogen, Karlsruhe, Germany). All plasmids were

kindly provided by C. J. Kirschning, Technical University Munich, Munich, Germany. After

4 h, medium was changed and cells were cultured in DMEM/10 % FCS over night.

Stimulation was performed in DMEM w/o FCS for 16 h. Cells were lysed and ß-galactosidase

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and luciferase activity was measured using a kit based on chemiluminescence (Roche,

Mannheim, Germany).

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), transfectants were additionally supplemented with

400 µg/ml G418 (Gibco, Eggenstein, Germany). For stimulation experiments, cells were

cultured at a density of 5 x 104 cells/well in 12 well tissue culture plates without G418 over

night. Cells were washed with Ham’s medium w/o FCS and transfection with expression

plasmids encoding for hTLR-2 (0,01 µg), hCD14 (0,01 µg), ß-galactosidase (0,1 µg) and the

ELAM NF-κB luciferase reporter plasmid (0,25 µg) for 16 h was performed employing 0,8

µl/well Fugene® (Roche, Mannheim, Germany). Cells were washed and stimulated with LPS

and LTA preparations in 1 ml Ham’s medium for 24 h followed by lysis and measurement of

ß-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 (Gibco, Eggenstein, Germany).

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 (Figure 1). LTA derived from staphylococci displays a faint staining pattern when

treated with silver staining reagents. Due to the higher carbohydrate content of PLTA it

exhibited a stronger staining pattern displaying two distinct major bands of approximately 31

and 36 kDa in size which 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 to 40 kDa range (Fig. 1

C). 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 immuno-detection method.

Induction of TNF-α 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

(PBMC) in the absence of serum. Both, PLTA and SLTA, induced TNF-α in a dose

dependent manner without substantial differences in the activation profiles (Fig. 2 A). As

compared to LPS, cytokine levels induced by LTA were lower, and under serum-free

conditions LTA had to be used at concentrations in the range up to 3 orders of magnitude

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greater as compared to LPS to elicit comparable levels of TNF-α. 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-α release when

monocytes were preincubated with MDP at 100 ng/ml as compared to LTA alone (Fig. 2 B

and C).

Effect of Serum, Recombinant Human LPS-Binding Protein (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 towards

monocytes in a comparable manner as well documented for LPS (12,13) we hypothesized that

LBP may be one of the major serum components mediating this effect for LTA, too. When

PBMCs were stimulated with PLTA and SLTA in the presence of LBP-concentrations

ranging from 0.1 to 10 µg/ml, significant enhancing effects on cytokine induction by both

LTAs were detected (Fig. 3 C). 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-α 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 less increased degree of

monocyte activation. A similar activity profile was obtained for the enhancing effects of LBP

in LPS-induced TNF-α release that was found to be maximal a the lowest LBP concentration

tested and less pronounced at higher LBP concentrations of 1 and 10 µg/ml, respectively (Fig.

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3 C). 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. 4 A), and for PLTA the same characteristics were observed (Fig. 4 B). LPS, in contrast

to LTA, stimulated cells independently of CD14 when higher concentrations were tested (Fig.

4 C). Comparable results were obtained when blocking effects were investigated in the

presence of serum instead of recombinant LBP (Fig. 4 D).

Gel-Shift Analysis on the Interaction of LTA with sCD14 and LBP

As the data obtained from the 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 towards higher

mobility was observed, combined with the additional detection of a slower migrating band

(Fig. 5 A). 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) as

compared to 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. 5 B). 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 as compared to 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 towards higher mobility (Fig. 5 AB, lanes 7) after

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incubation with PLTA and SLTA. Here, LBP-mediated catalytic transfer of LPS towards

sCD14 was also observed (Fig. 5 C), consistent with previous results from others (56).

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, in addition transiently transfected with human MD-2 and combinations of MD-

2 with TLR-2 or TLR–4. In this system, PLTA and SLTA turned out to act as ligands of TLR-

2 also as only transfection with TLR-2 led to enhanced activation of the cells when stimulated

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

due to a point mutation while 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 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). Over-expression

of CD14 in addition to TLR-2 in wild-type CHO cells significantly enhanced the TLR-2-

dependent activation of NF-κB by PLTA and SLTA confirming the involvement of CD14 in

LTA recognition (Fig. 6 F).

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 a MD-2 dependent cellular activation by LTA via TLR-2 and -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

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detect any interaction of our LTA preparations with TLR-4, hence we tried to determine

whether MD-2 had any effect on the translocation of NF-κB 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 the PLTA and SLTA NF-κB 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. 7 B), 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. 7 C). Transient transfection with

human TLR-2 restored LTA-mediated activation, but additional transfection of human MD-2

failed to increase the NF-κB translocation caused by LTA in line with the results obtained for

HEK293/CD14 cells.

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Discussion

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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 immuno-stimulatory 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 lately 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

to 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

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Discussion

18

as well as the presence of bands being 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 to LPS, LTA preparations by

concentration were less active, however, the used concentrations of LTA (1 µg~107 CFU) as

well as of LPS (20 ng~107 CFU) are comparable in orders of magnitude 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 TNFα-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). 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

towards 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 studies on outer membrane

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Discussion

19

glycolipids of Treponema spp. sharing structural similarities with LTA (64,65). Since all

these molecules differ greatly regarding 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 to the uncomplexed protein, indicating an interaction between these structures. The

addition of LTA to LBP also induced an increased electropheretic 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 an

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.

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Discussion

20

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-κB 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. Due to these

observations, we are furthermore 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 in part 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 from 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.

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References

21

References

1. Medzhitov, R., and Janeway, C. A., Jr. (1997) Cell 91, 295-298

2. Munford, R. S., and Pugin, J. (2001) Am. J. Respir. Crit. Care Med. 163, 316-321

3. Pfaller, M. A., Jones, R. N., Doern, G. V., and Kugler, K. (1998) Antimicrob. Agents

Chemother. 42, 1762-1770

4. Fluit, A. C., Schmitz, F. J., and Verhoef, J. (2001) Eur. J. Clin. Microbiol. Infect. Dis.

20, 188-191

5. Greenwood, B. (1999) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 354, 777-785

6. Diekema, D. J., Pfaller, M. A., Jones, R. N., Doern, G. V., Kugler, K. C., Beach, M.

L., and Sader, H. S. (2000) Int. J. Antimicrob. Agents 13, 257-271

7. Metlay, J. P. (2002) Curr. Opin. Infect. Dis. 15, 163-167

8. Glauser, M. P., Zanetti, G., Baumgartner, J. D., and Cohen, J. (1991) Lancet 338, 732-

736

9. Rietschel, E. T., Brade, H., Holst, O., Brade, L., Muller-Loennies, S., Mamat, U.,

Zahringer, U., Beckmann, F., Seydel, U., Brandenburg, K., Ulmer, A. J., Mattern, T.,

Heine, H., Schletter, J., Loppnow, H., Schonbeck, U., Flad, H. D., Hauschildt, S.,

Schade, U. F., Di Padova, F., Kusumoto, S., and Schumann, R. R. (1996) Curr. Top.

Microbiol. Immunol. 216, 39-81

10. Alexander, C., and Rietschel, E. T. (2001) J. Endotoxin Res. 7, 167-202

11. Leeson, M. C., Fujihara, Y., and Morrison, D. C. (1994) Infect. Immun. 62, 4975-4980

12. Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison,

J. C., Tobias, P. S., and Ulevitch, R. J. (1990) Science 249, 1429-1431

13. Schumann, R. R., and Latz, E. (2000) Chem. Immunol. 74, 42-60

by guest on January 24, 2020http://w

ww

.jbc.org/D

ownloaded from

References

22

14. Kitchens, R. L. a. M., R.S. (1999) in Endotoxin in Health and Disease (Brade H., O.

S. M., Vogel S.N., Morrison D.C., ed), pp. 521, Marcel Dekker, Inc., New York, Basel

15. van Deventer, S. J. H., Pajkrt, D. (1999) in Endotoxin in Health and Disease (Brade

H., O. S. M., Vogel S.N., Morrison D.C., ed), pp. 379, Marcel Dekker, Inc., New

York, Basel

16. Goyert, S. M., Ferrero, E., Rettig, W. J., Yenamandra, A. K., Obata, F., and Le Beau,

M. M. (1988) Science 239, 497-500

17. Haziot, A., Chen, S., Ferrero, E., Low, M. G., Silber, R., and Goyert, S. M. (1988) J.

Immunol. 141, 547-552

18. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J., and Mathison, J. C. (1990)

Science 249, 1431-1433

19. Gay, N. J., and Keith, F. J. (1991) Nature 351, 355-356

20. Means, T. K., Golenbock, D. T., and Fenton, M. J. (2000) Cytokine Growth Factor

Rev. 11, 219-232

21. Janeway, C. A., Jr., and Medzhitov, R. (2002) Annu. Rev. Immunol. 20, 197-216

22. Beutler, B., Du, X., and Poltorak, A. (2001) J. Endotoxin Res. 7, 277-280

23. Shimazu, R., Akashi, S., Ogata, H., Nagai, Y., Fukudome, K., Miyake, K., and

Kimoto, M. (1999) J. Exp. Med. 189, 1777-1782

24. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T.,

Kosugi, A., Kimoto, M., and Miyake, K. (2002) Nat. Immunol. 3, 667-672

25. Heumann, D., Barras, C., Severin, A., Glauser, M. P., and Tomasz, A. (1994) Infect.

Immun. 62, 2715-2721

26. Dziarski, R., Jin, Y. P., and Gupta, D. (1996) J. Infect. Dis. 174, 777-785

27. Gupta, D., Wang, Q., Vinson, C., and Dziarski, R. (1999) J. Biol. Chem. 274, 14012-

14020

by guest on January 24, 2020http://w

ww

.jbc.org/D

ownloaded from

References

23

28. Schneider, O., Michel, U., Zysk, G., Dubuis, O., and Nau, R. (1999) Neurology 53,

1584-1587

29. Heer, C., Stuertz, K., Reinert, R. R., Mader, M., and Nau, R. (2000) Infection 28, 13-

20

30. van Langevelde, P., van Dissel, J. T., Ravensbergen, E., Appelmelk, B. J., Schrijver, I.

A., and Groeneveld, P. H. (1998) Antimicrob. Agents Chemother. 42, 3073-3078

31. Bhakdi, S., Klonisch, T., Nuber, P., and Fischer, W. (1991) Infect. Immun. 59, 4614-

4620

32. Keller, R., Fischer, W., Keist, R., and Bassetti, S. (1992) Infect. Immun. 60, 3664-

3672

33. De Kimpe, S. J., Kengatharan, M., Thiemermann, C., and Vane, J. R. (1995) Proc.

Natl. Acad. Sci. U S A 92, 10359-10363

34. Yang, S., Tamai, R., Akashi, S., Takeuchi, O., Akira, S., Sugawara, S., and Takada, H.

(2001) Infect. Immun. 69, 2045-2053

35. Dziarski, R., Wang, Q., Miyake, K., Kirschning, C. J., and Gupta, D. (2001) J.

Immunol. 166, 1938-1944

36. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., and Kirschning, C. J. (1999) J.

Biol. Chem. 274, 17406-17409

37. Takeuchi, O., Hoshino, K., and Akira, S. (2000) J. Immunol. 165, 5392-5396

38. Ginsburg, I. (2002) Lancet Infect. Dis. 2, 171-179

39. Gao, J. J., Xue, Q., Zuvanich, E. G., Haghi, K. R., and Morrison, D. C. (2001) Infect.

Immun. 69, 751-757

40. Morath, S., Geyer, A., Spreitzer, I., Hermann, C., and Hartung, T. (2002) Infect.

Immun. 70, 938-944

41. Morath, S., Geyer, A., and Hartung, T. (2001) J. Exp. Med. 193, 393-397

by guest on January 24, 2020http://w

ww

.jbc.org/D

ownloaded from

References

24

42. Morath, S., Stadelmaier, A., Geyer, A., Schmidt, R. R., and Hartung, T. (2002) J. Exp.

Med. 195, 1635-1640

43. Fischer, W. (1994) Med. Microbiol. Immunol. (Berl.) 183, 61-76

44. Greenberg, J. W., Fischer, W., and Joiner, K. A. (1996) Infect. Immun. 64, 3318-3325

45. Fischer, W., Mannsfeld, T., and Hagen, G. (1990) Biochem. Cell Biol. 68, 33-43

46. Behr, T., Fischer, W., Peter-Katalinic, J., and Egge, H. (1992) Eur. J. Biochem. 207,

1063-1075

47. Yoshimura, A., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R., and Golenbock,

D. (1999) J. Immunol. 163, 1-5

48. Echchannaoui, H., Frei, K., Schnell, C., Leib, S. L., Zimmerli, W., and Landmann, R.

(2002) J. Infect. Dis. 186, 798-806

49. Holtje, J. V., and Tomasz, A. (1975) J. Biol. Chem. 250, 6072-6076

50. Tsai, C. M., and Frasch, C. E. (1982) Anal. Biochem. 119, 115-119

51. Cosenza, H., Quintans, J., and Lefkovits, I. (1975) Eur. J. Immunol. 5, 343-349

52. Michelsen, K. S., Aicher, A., Mohaupt, M., Hartung, T., Dimmeler, S., Kirschning, C.

J., and Schumann, R. R. (2001) J. Biol. Chem. 276, 25680-25686

53. Hamann, L., Schumann, R. R., Flad, H. D., Brade, L., Rietschel, E. T., and Ulmer, A.

J. (2000) Eur. J. Immunol. 30, 211-216

54. Schromm, A. B., Lien, E., Henneke, P., Chow, J. C., Yoshimura, A., Heine, H., Latz,

E., Monks, B. G., Schwartz, D. A., Miyake, K., and Golenbock, D. T. (2001) J. Exp.

Med. 194, 79-88

55. Holtje, J. V., and Tomasz, A. (1975) Proc. Natl. Acad. Sci. U S A 72, 1690-1694

56. Hailman, E., Lichenstein, H. S., Wurfel, M. M., Miller, D. S., Johnson, D. A., Kelley,

M., Busse, L. A., Zukowski, M. M., and Wright, S. D. (1994) J. Exp. Med. 179, 269-

277

by guest on January 24, 2020http://w

ww

.jbc.org/D

ownloaded from

References

25

57. Opitz, B., Schröder, N. W., Spreitzer, I., Michelsen, K. S., Kirschning, C. J.,

Hallatschek, W., Zähringer, U., Hartung, T., Göbel, U. B., and Schumann, R. R.

(2001) J. Biol. Chem. 276, 22041-22047

58. Heine, H., Kirschning, C. J., Lien, E., Monks, B. G., Rothe, M., and Golenbock, D. T.

(1999) J. Immunol. 162, 6971-6975

59. Manthous, C. A., Hall, J. B., and Samsel, R. W. (1993) Chest 104, 1872-1881

60. Ingraham, J. M., Maaloe, O., and Neidhart, F. C. (1983) in Growth of the bacterial

cell, pp. 3, Sinauer Associates Inc., Sunderland, MA

61. Wolfert, M. A., Murray, T. F., Boons, G. J., and Moore, J. N. (2002) J. Biol. Chem.

277, 39179-39186

62. Fan, X., Stelter, F., Menzel, R., Jack, R., Spreitzer, I., Hartung, T., and Schütt, C.

(1999) Infect. Immun. 67, 2964-2968

63. Hermann, C., Spreitzer, I., Schröder, N. W., Morath, S., Lehner, M. D., Fischer, W.,

Schutt, C., Schumann, R. R., and Hartung, T. (2002) Eur. J. Immunol. 32, 541-551

64. Schröder, N. W., Opitz, B., Lamping, N., Michelsen, K. S., Zähringer, U., Göbel, U.

B., and Schumann, R. R. (2000) J. Immunol. 165, 2683-2693

65. Schröder, N. W., Pfeil, D., Opitz, B., Michelsen, K. S., Amberger, J., Zähringer, U.,

Göbel, U. B., and Schumann, R. R. (2001) J. Biol. Chem. 276, 9713-9719

66. Lamping, N., Dettmer, R., Schröder, N. W., Pfeil, D., Hallatschek, W., Burger, R., and

Schumann, R. R. (1998) J. Clin. Invest. 101, 2065-2071

67. Zweigner, J., Gramm, H. J., Singer, O. C., Wegscheider, K., and Schumann, R. R.

(2001) Blood 98, 3800-3808

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Footnotes

26

Footnotes

1 This work was supported in part by grants given by the Bundesministerium für Bildung

und Forschung (BMBF) to R.R.S. (Capnetz), by Clinique La Prairie Research to C.A, and

the Deutsche Forschungsgemeinschaft (DFG) to R.R.S. and N.W.J.S. (Innate Immunity) as

well as T.H. (Innate Immunity), respectively

2 Address correspondence and reprint requests to:

Ralf R. Schumann, M.D.

Institut für Mikrobiologie und Hygiene

Universitätsklinikum Charité, Humboldt-Universität zu Berlin

Dorotheenstr. 96, D-10117 Berlin, Germany.

Tel.: +49-30-450-524141

FAX: +49-30-450-524904

e-mail: ralf.schumann@charite.de

3 Abbreviations used in this paper: LBP, LPS binding protein; LPS, lipopolysaccharide;

LTA, lipoteichoic acid; MDP, muramyldipeptide; NF-κB, nuclear factor κB; PBMC:

peripheral blood mononuclear cells; PAGE, polyacrylamide gel electrophoresis; PG,

peptidoglycan; PLTA, LTA derived from S. pneumoniae; sCD14, soluble CD14; SLTA,

LTA derived from S. aureus; TLR, toll like receptor, TNF-α, tumor necrosis factor α

4 We acknowledge the excellent technical assistance of Fränzi Creutzburg, Nicole

Siegemund (Berlin), Nina Grohmann, Katharina Jacob (Borstel) and Leo Cobianchi

(Konstanz).

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Figure Legends

27

Figure Legends

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).

Fig. 2: Induction of TNF-α 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-α was estimated after 4 h by ELISA. B, 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-α was

estimated after 4 h by ELISA. Experiments were performed in duplicates. Shown are

representatives out of 3 experiments.

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-α 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

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28

recombinant human LBP. Experiments were performed in duplicates (A to C) and

quadruplicates (D). Shown are representatives out of 3 experiments.

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

to C) or triplicates (D). Shown are representatives out of 3 experiments.

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, and the

modulating effects of LBP on complexation of LTA and sCD14 were analyzed by native

PhastGel™ 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 % PhastGels™ and subsequent silver staining employing the PhastSystem™ 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 susequently

adjusted to a final concentration of 2 % (w/v) SDS and additionally analyzed by PhastGel™

SDS electrophoresis and silver staining (lower panels).

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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 ß-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 ß-galactosidase and luciferase activity was estimated.

Activation is indicated using arbitrary units (ß-galactosidase/luciferase x 10). CHO/CD14

cells were transiently transfected with plasmids encoding for ß-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 Effectene®, followed by stimulation with PLTA, SLTA (500 ng/ml

each) or LPS (S. minnesota, 100 ng/ml) for 16 h. Activation of cells was estimate as

described above. F: CHO wild-type cells were transiently transfected with a plasmid

encoding ß-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 out of 3 experiments.

Fig. 7: Effect of MD-2 on Activation of HEK293 cells by LTA

HEK293/CD14 cells were transiently transfected with plasmids encoding for ß-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 out of 3 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, ß-galactosidase and ELAM for 24 h

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30

employing Effectene®. 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 to the controls. Experiments were performed in triplicates, shown is one

representative out of 2 experiments.

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Figures

31

Fig.1

Fig. 1

3.0

6.0

14.4

21.531.036.5

55.4

66.3

LPS SLTA PLTA PLTA

A C

3.06.0

14.4

21.531.036.5

55.4

66.3

LPS SLTA PLTA

BSilver-stain Colloidal Gold α-TEPC-15

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Figures

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Fig. 2

hTNF (ng/ml)

SLTAPLTALPS

0

1

2

3

4

5

6

7

0.00010.001 0.01 0.1 1 10

A

concentration of stimuli (µg/ml)

0

1

2

3

4

5

6

0 0.04 0.2 1

+ MDP 100 ng/mlw/o MDP

hTNF (ng/ml)B

concentration of SLTA (µg/ml)

concentration of PLTA (µg/ml)

0

1

2

3

4

5

6

0 0.04 0.2 1

+ MDP 100 ng/mlw/o MDP

hTNF (ng/ml)C

Fig.2

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Fig.3

Fig. 3

0

0.5

1

1.5

2

2.5

3

3.5

none PLTA2 µg/ml

PLTA0.4 µg/ml

SLTA2 µg/ml

SLTA0.4 µg/ml

w/o LBP0,1 µg/ml LBP1 µg/ml LBP10 µg/ml LBP

0

5

10

15

20

25

30

LPS20 ng/ml

ChTNF(ng/ml)

0

5

10

15

20

25

30

0.1 1 10

w/o serum+ 2.5 % serum

concentration of PLTA (µg/ml)

02

6

10

14

18

0.1 1 10

w/o serum+ 2.5 % serum

concentration of SLTA (µg/ml)

AhTNF(ng/ml)

BhTNF(ng/ml)

****

**

****

*

** *

**** *

**

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Fig.4

0

0.4

0.8

1.2

1.6

2

0.08 0.4 2 10

+ LBP+ LBP + My4

0

0.4

0.8

1.2

1.6

2

0.08 0.4 2 10

+ LBP+ LBP + My4

0

2

4

6

8

10

0.08 0.4 2 10

+ LBP+ LBP + My4

PLTA2 µg/ml

SLTA2 µg/ml

LPS4 ng/ml

0

2

4

6

8

10

12

2 %Serum w/o My42 %Serum +My4

none

Fig. 4

A

concentration of SLTA (µg/ml) concentration of PLTA (µg/ml)

hTNF(ng/ml)

BhTNF(ng/ml)

concentration of LPS (ng/ml)

ChTNF(ng/ml)

DhTNF(ng/ml)

****

**

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Figures

35

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Figures

36

Fig. 6

2

6

10

14

none PLTA SLTA LPS

w/o CD14+ CD14

rel. light units

F

2

6

10

14

none PLTA SLTA LPS

rel. light units

A

2

6

10

14

none PLTA SLTA LPS

rel. light units

C

2

6

10

14

none PLTA SLTA LPS

rel. light units

B

MD-2

TLR-2+ MD-2

TLR-4+ MD-2

2

6

10

14

none PLTA SLTA LPS

rel. light units

D

MD-2

2

6

10

14

none PLTA SLTA LPS

rel. light units

E

TLR-2+ MD-2

TLR-2+ MD-2

CHO (Wild-type)

HEK293/CD14 CHO/CD14

** **

**

** ** **

**

* *

*

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Figures

37

Fig. 7

A

05

101520253035

0 2 10 50

rel. light units

amount of MD-2 transfected (ng)

0

10

20

30

40

50

0 2 10 50

rel. light units

B

amount of MD-2 transfected (ng)

none+ TLR-2+ TLR-2+ MD-2

0

1

2

3

4

PLTA2 µg/ml

SLTA2 µg/ml

fold increaseof light units

C

Fig.10

** **

SLTA 1 µg/ml PLTA 1 µg/mlnone

LPS 200 ng/ml none

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Hartung, Ulrich Zähringer, Ulf B. Göbel, Joerg R. Weber and Ralf R. SchumannNicolas W.J. Schröder, Siegfried Morath, Christian Alexander, Lutz Hamann, Thomas

MD-2 are not involvedtoll-like receptor (TLR)-2, LPS binding protein (LBP) and CD14 while TLR-4 and Lipoteichoic acid (LTA) of S. pneumoniae and S. aureus activates immune cells via

published online February 19, 2003J. Biol. Chem. 

  10.1074/jbc.M212829200Access the most updated version of this article at doi:

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