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Originally published In Press as doi:10.1074/jbc.M605966200 on August 23, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31448-31456, October 20, 2006
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Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein*

Mareike Mueller{ddagger}, Cordula Stamme§, Christian Draing||, Thomas Hartung||, Ulrich Seydel{ddagger}, and Andra B. Schromm**1

From the Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Divisions of {ddagger}Biophysics, §Cellular Pneumology, and **Immunobiophysics, Department of Immunochemistry and Biochemical Microbiology, D-23845 Borstel, Germany, the ||University of Konstanz, Department of Biochemical Pharmacology, D-78457 Konstanz, Germany, and the University of Lübeck, Department of Anaesthesiology, D-23538 Lübeck, Germany

Received for publication, June 22, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor {alpha} under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor {alpha} and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The key to a successful pathogen defense is the recognition of pathogen-associated molecular structures by receptors of the innate immune system leading to a proinflammatory response. Systemic production of proinflammatory mediators, however, can also lead to sepsis, a complex clinical syndrome caused by an overshooting host response (1). According to epidemiological studies, infections by Gram-positive bacteria are responsible for about half of the cases of systemic infections in the United States and Europe, and Staphylococcus aureus is the most frequently isolated Gram-positive pathogen in invasive infections and trauma patients (2, 3).

For Gram-negative bacteria, the pathogenesis of septic shock has been attributed to the activation of the innate immune system by lipopolysaccharide (LPS)2 from the outer leaflet of the outer membrane of the organisms (4). Cell activation by LPS is mediated by a complex receptor cluster consisting of Toll-like receptor (TLR) 4 and MD-2 (5–8) and a number of further proteins such as CD11/CD18, CD55, CD81, GDF5, and CXCR4 (9–11) and the ion channel MaxiK (12, 13). Because of its amphiphilic structure, LPS forms supramolecular aggregates in an aqueous environment. The supramolecular structure of these aggregates critically determines the biological activity of LPS (14, 15). Transport of LPS aggregates to signaling proteins on the surface of mononuclear phagocytes represents the first step in cell activation. Multiple transport pathways for LPS exist to target host cells, including the transport of LPS in the serum by the soluble acute phase serum protein LPS-binding protein (LBP) (16, 17) or the soluble CD14 (sCD14) receptor (18) to the membrane-bound CD14 (mCD14) (19). However, only recently it has been shown that LBP also assumes a transmembrane configuration (mLBP) and in this configuration incorporates LPS aggregates into the cell membrane (20–22). Other reports show complex vectorial transport chains for LPS monomers (23).

In contrast to the well investigated receptor system for LPS from Gram-negative bacteria, little is known about the molecular mechanisms involved in the recognition of Gram-positive pathogens. Several studies suggest that also molecules of the cell wall mediate inflammatory responses of the host, namely peptidoglycan of the murein layer and lipoteichoic acid (LTA). LTA is an amphiphilic molecule anchored to the outer surface of the cytoplasmic membrane by a glycerolipid. Peptidoglycan and LTA are released from the cell wall during growth and especially under antibiotic treatment, and both molecules have been shown to express immune stimulatory activity (24, 25). After a long controversy about the immune stimulatory capacity and purity of commercial LTA preparations, establishment of a new purification protocol for LTA based on butanol extraction revealed that pure, LPS-free preparations of LTA from S. aureus exhibit immune stimulatory activity in human whole blood in high concentrations (25). LTA derived by this purification protocol was shown to be composed of a diacylglycerol lipid anchor covalently linked to a gentiobiose and a polymeric backbone that in S. aureus is composed of a central 1–3-linked glycerophosphate chain substituted with D-alanine, {alpha}-D-N-acetylglucosamine, and to a lesser extent unsubstituted 1–3-linked glycerophosphate residues. The immune stimulatory capacity of LTA could be confirmed by chemical synthesis of the lipid anchor containing a backbone of six glycerophosphate units carrying D-alanine and N-acetylglucosamine as substituents (26). However, the structural basis of cell activation by molecules derived from the Gram-positive cell wall is not conclusively defined. Although the molecular structures leading to activation of NOD proteins by peptidoglycan have recently been identified (27, 28), the molecular basis of its TLR2 activity is not fully understood (29, 30). Also, recent data indicate that alanine substitution of LTA might not be required for its biological activity (31).

The mechanisms of cell activation by LTA today are not understood. However, it is now generally accepted that LTA activates immune responses via a TLR2/TLR6 heterodimer (31, 32). There are, nevertheless, still conflicting reports on the requirement of CD14 (33, 34) and LBP in LTA-mediated cellular responses, ranging from enhancing to no effects of LBP on cell activation by LTA (32, 35, 36). In the current study, we investigated the role of serum and LBP in cell activation by LTA. Our data show that LTA interacts with LBP and that cell activation is strongly attenuated by this interaction. We present evidence for a differential regulation of LTA recognition by macrophages dependent on the absence and presence of LBP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Deep rough mutant LPS (Re LPS) was extracted from Salmonella enterica sv. Minnesota strain R595 according to the phenol/chloroform/petrol ether procedure (37). The LPS preparation was lyophilized and used in the natural salt form. The chemical purity of the LPS preparation was confirmed by mass spectrometry. Highly purified lipoteichoic acid was isolated from S. aureus as described previously (25). LPS and LTA were suspended in phosphate-buffered saline (Biochrom, Berlin, Germany) by thorough vortexing. The suspensions were temperature cycled at least twice between 4 and 56 °C, with each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. Suspensions were aliquoted and stored at –20 °C. Phosphatidylserine (PS) from bovine brain was purchased from Avanti-Polar Lipids (Alabaster, AL) and used without further purification. The fluorescent dyes N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE (NBD-PE) and N-(rhodamine B sulfonyl)-PE (Rh-PE) were purchased from Molecular Probes (Eugene, OR). Recombinant human LBP (456-amino acid holoprotein rLBP50) in 10 mM HEPES, pH 7.5, was a kind gift of XOMA LLC (Berkeley, CA). The monoclonal mouse anti-mouse LBP antibody biG33 cross-reacting with human LBP and the monoclonal anti-human CD14 antibody biG 14 were obtained from Biometec (Greifswald, Germany). Isotype-matched IgG1 control antibody was obtained from BD Biosciences (Heidelberg, Germany). PI-PLC was from Sigma.

Preparation of Macrophages and Incubation Conditions—Monocytes were isolated from human peripheral blood of healthy donors by the Hypaque-Ficoll gradient method and cultivated at 37 °C and 6% CO2 in Teflon bags in RPMI 1640 medium (endotoxin ≤0.01 EU/ml; Biochrom) containing 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 4% heat-inactivated human serum type AB from healthy donors. The cells were cultured in the presence of 2 ng/ml macrophage colony-stimulating factor for 7 days to differentiate monocytes to macrophages. To determine cytokine induction after cell stimulation, the cells were seeded at 200-µl aliquots of a suspension of 1 x 106 cells/ml in 96-well tissue culture dishes (Nunc, Wiesbaden, Germany) in RPMI 1640 medium containing 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, with or without 4% human serum. Cell-free supernatants were collected 4 h after stimulation for TNF{alpha} determination or 24 h after stimulation for IL-6 and IL-8 determination, respectively, and stored at –20 °C until determination of cytokine content. The data shown are the means and standard deviations (±S.D.) of triplicate samples of one experiment and representative of at least three independent experiments.

Alveolar macrophages of the rat were isolated by lung lavage of male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) as described (38). Cell viability was checked by erythrosin B exclusion and routinely averaged 94–98%. The cells were washed once in RPMI and resuspended in RPMI containing 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, with or without 10% heat-inactivated fetal calf serum (Linaris, Bettingen, Germany). To determine cytokine induction by LTA, the cells were seeded at 0.5 x 106 cells/well in 96-well tissue culture dishes and stimulated with LTA in the absence or presence of serum. To cleave cell-bound CD14 from the cell surface, the cells were treated with 0.5 unit/ml PI-PLC for 60 min at 37 °C prior to stimulation. Cell-free supernatants were harvested after 4 h of stimulation for the determination of TNF{alpha}.

Cytokine Determination—Human TNF{alpha} and human IL-6 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using monoclonal mouse antibody against human IL-6 and POD-conjugated rabbit anti-human IL-6 antibody and monoclonal mouse antibody against human TNF{alpha} and POD-conjugated rabbit anti-human TNF{alpha} antibody, respectively (Intex, Muttant, Switzerland) as stated in detail elsewhere (39). Rat TNF{alpha} and human IL-8 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using Cytosets from BIOSOURCE (Solingen, Germany) exactly according to the manufacturer's protocol. The data shown are the means ± S.D. of triplicate samples of one representative experiment.

Fluorescence Resonance Energy Transfer Spectroscopy—The fluorescence resonance energy transfer (FRET) technique was used as a probe dilution assay (17, 40) to obtain information on the intercalation of LBP and LTA into liposomes made from the negatively charged phospholipid PS. For the FRET experiments, liposomes were double-labeled with NBD-PE and Rh-PE in chloroform [PS]:[NBD-PE]:[Rh-PE] of 100:1:1 molar ratios. The solvent was evaporated under a stream of nitrogen, and the lipids were resuspended in phosphate-buffered saline, mixed thoroughly, and sonicated with a Branson sonicator for 1 min (1 ml of solution). Subsequently, the preparation was temperature-cycled at least twice between 4 and 56 °C, with each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. A preparation of 900 µl of the double-labeled liposomes (10–5 M) at 37 °C was excited at 470 nm (excitation wavelength of NBD-PE), and the intensities of the emission light of the donor NBD-PE (531 nm) and acceptor Rh-PE (593 nm) were measured simultaneously on the fluorescence spectrometer SPEX F1T11 (SPEX Instruments, Edison, NY). LBP (5,5 µg/ml) and LTA aggregates (30 µg/ml) were added to liposomes after 50 and 100 s, respectively. Because FRET spectroscopy is used here as a probe dilution assay, intercalation of unlabeled molecules such as LBP or LTA causes an increase of the distance between donor and acceptor and thus leads to a reduced energy transfer. This again causes an increase of the donor and a decrease of the acceptor intensities. For a qualitative analysis of experiments, the quotient of the intensities of the donor dye and the acceptor dye are plotted against time (denoted in the following as the FRET signal). The data shown are representative for three independent experiments.

Plasmon Resonance Spectroscopy—A surface plasmon resonance technique (41) was used as a binding assay to detect interaction of LBP and LPS with immobilized liposomes made from PS. First, L1 sensor chip (Biacore AB, Uppsala, Sweden) was pretreated with a 10 µM suspension of PS liposomes to obtain an immobilized lipid matrix for interaction experiments with LBP and LTA. LBP and LTA were added at concentrations of 10 µM and 100 nM, respectively. The running buffer was phosphate-buffered saline at pH 7.0, and the experiments were performed at 37 °C at a flow rate of 10 µl/min in a BIACORE 3000. The data are presented as response units in dependence on time for one representative experiment of a total of three.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Activation of Human Macrophages by LTA Is Attenuated in the Presence of Serum—We tested the potential immune stimulatory activity of LPS-free LTA preparations using human macrophages that were in vitro differentiated from peripheral blood mononuclear cells. Stimulation of macrophages with LTA in the presence of 4% human serum led to a dose-dependent production of the proinflammatory cytokine TNF{alpha} (Fig. 1). Under these conditions, 3–10 µg/ml LTA were required to induce TNF{alpha} production. Compared with the amount of TNF{alpha} induced by the same cells after stimulation with 1 ng/ml LPS under serum-free conditions (1046 ± 24 pg TNF{alpha}/ml), the amounts of TNF{alpha} induced by these high concentrations of LTA were about three times lower (335 ± 7pgTNF{alpha}/ml induced by 10 µg/ml LTA). However, stimulation of the cells with LTA under serum-free culture conditions led to a significant enhancement of the cytokine production. Under these conditions, high concentrations of LTA induced similar amounts of TNF{alpha} (1002 ± 48 pg TNF{alpha}/ml induced by 1 µg/ml LTA) as induced by 1 ng LPS. In contrast to serum-containing conditions, TNF{alpha} production was already induced by 30 ng/ml LTA under serum-free conditions and increased dose-dependently with increasing concentrations of LTA, reaching saturation of cell activation at 300 ng/ml LTA. Similar results could be observed for the late pro-inflammatory mediators IL-6 and IL-8, both of which showed a dose-dependent inhibition in the presence of serum after 24 h of stimulation (Fig. 2). These results suggest that a compound in serum strongly attenuates cell activation by LTA.


Figure 1
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  		FIGURE 1.
Cell activation of human macrophages by LTA is strongly attenuated in the presence of serum. Human blood macrophages were stimulated in the absence or presence of 4% AB serum with the indicated concentrations of LTA or LPS. Cell-free supernatants were harvested after 4 h for the determination of TNF{alpha}. Cytokine concentrations are given as means ± S.D. of triplicate samples. In unstimulated cells, TNF{alpha} was not detectable. The data shown are representative of five independent experiments. N.D., not detectable.

 
LTA Interacts with LBP and Inhibits the Integration of LBP into Phospholipid Membranes—LBP is an important serum protein involved in the innate immune recognition of a variety of pathogen-associated molecules. It has been shown to interact with LPS and enhance cell activation at low concentrations of LPS. However, at high concentrations of LBP as they appear in acute phase serum, inhibitory effects of LBP on cellular responses to LPS have been reported (21, 42, 43). Because LBP has been found to interact also with other virulence factors (44–46), we postulated that LBP may be the component in serum modulating cellular response to LTA. We used a liposome assay based on fluorescence resonance energy transfer to gain information on the interaction of LTA with LBP. We have shown in previous reports that LBP is not only a soluble protein but readily intercalates into liposomal membranes (20, 21). This hydrophobic interaction of LBP with phospholipid membranes is markedly enhanced for negatively charged phospholipids. In the presence of LPS, LBP mediates a transport of LPS into the phospholipid bilayer of liposomes (17). We used this liposome assay to investigate the influence of LTA on the interaction of LBP with liposomal membranes. When recombinant human LBP is added to liposomes composed of the negatively charged phospholipid phosphatidylserine, it readily intercalates into the phosholipid bilayer as can be taken from the increase in the FRET signal (ratio of donor and acceptor signals; Fig. 3). When LTA is added to the liposomes in the absence of LBP, no change in the FRET signal could be observed, indicating that LTA does not intercalate into the phospholipid membrane spontaneously. Additional application of LBP to the mixture of liposomes and LTA did not lead to any changes in the FRET signal.


Figure 2
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  		FIGURE 2.
Stimulation of late pro-inflammatory mediators by LTA in human macrophages is much more sensitive under serum-free conditions. Human blood macrophages were stimulated in the absence (left panel) or presence (right panel) of 4% AB serum with the indicated concentrations of LTA. Cell-free supernatants were harvested after 24 h for the determination of IL-6 (A and B) and IL-8 (C and D). Cytokine and chemokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.

 


Figure 3
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  		FIGURE 3.
LTA interacts with LBP and inhibits the integration of LBP into phospholipid membranes. Time kinetics of changes in the FRET signal from double-labeled PS-liposomes (10–5 M) upon subsequent addition of phosphate-buffered saline (gray curve a) or LTA (black curve b) (30 µg/ml) and LBP (5.5 µg/ml). Time points of addition of the agents are marked as steps 1 and 2. The data shown are representative of three independent experiments.

 
LTA Does Not Interact with Negatively Charged Phospholipid Membranes in the Absence or Presence of LBP—To further investigate the interaction of LTA and LBP with phospholipid membranes, we employed plasmon resonance spectroscopy. This method provides information on the binding of different partners in a flow-through system. We coated phosphatidylserine liposomes onto the surface of the flow-through chip and subsequently added LTA and LBP. As can be seen from Fig. 4, the addition of LTA did not lead to an increase of response units (time point 3), indicating that LTA did not bind to the phospholipid membranes. When LBP was added (time point 4), binding of LBP to the phospholipid membrane resulted in an increase in response units. Because the experiments were performed under continuous flow-through conditions, LTA was not present at the time of addition of LBP, explaining the absence of any inhibitory effects of LTA on the interaction of LBP with the membrane. In accordance to the results observed in the liposome assay, the addition of LTA to membrane-bound LBP (time points 6 and 7) did not lead to an increase of response units. These results confirm that LTA does not bind to phospholipid membranes in the absence or presence of membrane-associated LBP. In addition, there is no indication for any binding of LTA to membrane-associated LBP.

LBP Inhibits LTA-induced Cytokine Production from Human Macrophages—To gain more information on the role of LBP in the process of cell activation by LTA, we titrated recombinant LBP to human macrophages stimulated with LTA under serum-free conditions. The production of the early pro-inflammatory cytokine TNF{alpha} clearly showed a dose-dependent inhibition of cytokine production by LBP after 4 h of stimulation (Fig. 5). The inhibitory effects of LBP were observed already at 100 ng/ml. These LBP concentrations are even lower than the amount of LBP present in normal human serum (1–10 µg/ml). Thus, LBP appears to be a negative regulator of cell activation by LTA at physiological concentrations.

Cell Activation by LTA Is Modulated by Soluble LBP, but Not by Cell-associated mLBP—To elucidate the role of the different forms of LBP: soluble sLBP and membrane-bound mLBP, we used inhibitory antibodies to LBP to neutralize the effects of soluble or cell-associated protein. Human macrophages were washed several times to remove soluble LBP and stimulated under serum-free conditions with LTA, and TNF{alpha} production was determined. The addition of recombinant sLBP decreased the production of TNF{alpha} induced by LTA (Fig. 6). When sLBP was preincubated with an anti-LBP antibody, the inhibitory effect on cell stimulation was attenuated. In contrast, when cells were directly incubated with anti-LBP antibody in the absence of sLBP, no effect on cell activation was observed. Similar results were observed when cells were incubated with an isotype-matched control antibody. These results show that negative regulation of cell activation by LTA is due to interaction with soluble LBP, whereas mLBP does not appear to be involved in cell activation by LTA.


Figure 4
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  		FIGURE 4.
LTA does not interact with negatively charged phospholipid membranes in the absence or presence of LBP. PS liposomes (10 µM) were immobilized on the BIACore chip, and LBP and LTA were added at concentrations of 10 µM and 100 nM, respectively. Time points of addition of the agents are marked by numbers. The data are presented as response units in dependence on time for one representative experiment of a total of three.

 


Figure 5
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  		FIGURE 5.
LBP dose-dependently inhibits activation of human macrophages by LTA. Human macrophages from elutriated monocytes were incubated for 10 min in serum-free medium containing the indicated concentrations of recombinant human LBP and subsequently stimulated with LTA. Cell-free supernatants were harvested after 4 h for the determination of TNF{alpha}. Cytokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.

 
Primary Alveolar Macrophages Show Sensitive Responses to LTA under Serum-free Culture Conditions—The inhibitory effects of LBP on cell activation by LTA described so far were all observed under experimental conditions where macrophages derived by in vitro differentiation of blood monocytes were stimulated under serum-free conditions. These culture conditions are likely to resemble conditions as they are given for tissue macrophages that are not in contact with serum and serum proteins. However, the experimental conditions might not completely resemble the in vivo conditions of tissue macrophages. Thus, we wanted to confirm our results with in vivo differentiated macrophages adapted to low serum concentrations or serum-free conditions. Alveolar macrophages are specialized cells of the pulmonary innate immune response to infections and are naturally adapted to very low serum or serum-free conditions in the lung under physiological conditions. We used alveolar macrophages derived from alveolar lavage of rats and stimulated these cells under serum-free and serum-containing conditions with LTA. As shown in Fig. 7, these cells show an increased sensitivity to low amounts of LTA under serum-free culture conditions, whereas the production of TNF{alpha} under serum-containing conditions is attenuated and resembles the dose response of in vitro differentiated macrophages derived from blood.


Figure 6
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  		FIGURE 6.
Cell activation by LTA is modulated by soluble LBP, whereas cell-associated mLBP has no effects on cell activation by LTA. Human blood macrophages were washed five times in serum-free medium to remove all of the soluble proteins and then suspended in serum-free medium to investigate the influence of mLBP on cell activation by LTA. To investigate the effects of soluble LBP on cell activation by LTA, recombinant human LBP (1 µg/ml) was added to the cells and incubated for 25 min before stimulation (bar b). To block soluble or membrane associated LBP, anti-LBP antibody (15 µg/ml; bar d) or an isotype-matched control IgG1 antibody (15 µg/ml; bar e) were added to the cells directly or preincubated for 25 min with sLBP (bar c) before addition to cells. Subsequently, the cells were stimulated with the indicated concentrations of LTA. Cell-free supernatants were harvested after 4 h for the determination of TNF{alpha}. Cytokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.

 
Cell Activation by LTA Is Enhanced by mCD14 in Human Macrophages and Rat Alveolar Macrophages—Cell activation by LPS depends on the presence of CD14 in serum (sCD14) and on the cell surface (mCD14). To elucidate the role of CD14 in LTA-induced cell activation of macrophages under serum-free culture conditions, we stimulated human macrophages with LTA in serum-free medium in the absence and presence of a neutralizing antibody against CD14. In the presence of the anti-CD14 antibody, the production of TNF{alpha} was clearly reduced, indicating that mCD14 participates in cell activation under serum-free conditions (Fig. 8A). Similar results were obtained on rat alveolar macrophages. Because of the lack of a rat reactive CD14 antibody, we used enzymatic cleavage of CD14 as an alternative approach to remove CD14 from the cell surface (19, 47). Treatment of the cells with phospholipase C strongly inhibited the production of TNF{alpha} (Fig. 8B), confirming a role for mCD14 also in primary rat macrophages.


Figure 7
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  		FIGURE 7.
Primary alveolar macrophages from rats show sensitive responses to LTA under serum-free culture conditions. Alveolar macrophages from rats were suspended in medium containing 10% fetal calf serum (A) or serum-free medium (B) and stimulated with the indicated concentrations of LTA. Cell-free supernatants were harvested after 4 h for the determination of TNF{alpha}. Cytokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Infections with Gram-positive pathogens play a major role in community-acquired and especially in hospital-acquired infections, among which S. aureus represents one of the most common Gram-positive pathogens (48, 49). During the last decades, the number of S. aureus strains resistant to antibiotics has steadily increased, causing major problems concerning therapy of these infections (50). The mechanisms of the host response to Gram-positive infections are not very well understood. A problem in investigating mechanisms of the innate immune defense against Gram-positive pathogens was the lack of defined and pure compounds from Gram-positive bacteria activating host responses. Two molecular structures from the Gram-positive cell wall have been identified to activate a protective host response: peptidoglycan and lipoteichoic acid. However, many of the results obtained with commercially available preparations have been scrutinized with the concern of LPS contaminations (51). Recently, muramyldipeptide has been identified as the active component in peptidoglycan preparations inducing cell activation via the intracellular protein NOD2 (28). The reported TLR2-dependent activity of peptidoglycan preparations is still controversial (29, 30).


Figure 8
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  		FIGURE 8.
Cell activation of human macrophages and primary alveolar macrophages from rats by LTA depends on mCD14. A, human blood macrophages were washed five times in serum-free medium and then suspended in serum-free medium. To investigate the role of mCD14 on cell activation by LTA, anti-CD14 antibody (10 µg/ml; black bars) or an isotype-matched control IgG1 antibody (15µg/ml; hatched bars) were added to the cells and incubated for 25 min. Subsequently, the cells were stimulated with the indicated concentrations of LTA. B, alveolar macrophages from rats were suspended in serum-free medium, treated with 0.5 unit/ml PI-PLC for 60 min at 37 °C to cleave cell bound CD14, and then stimulated with the indicated concentrations of LTA. Cell-free supernatants were harvested after 4 h for the determination of TNF{alpha}. Cytokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.

 
To exclude contaminations, we first tested our LTA preparations for TLR dependence. LTA has been shown to activate immune responses via a TLR2/TLR6 heterodimer (31, 32). Using HEK293 cells stably expressing TLR2 and TLR4/MD-2, we could confirm that our LTA preparation was strictly dependent on TLR2 and did not show any TLR4 activity as shown by stimulation of interleukin 8 (data not shown), excluding contamination of LTA by LPS. Using these LPS-free preparations of LTA, we show here that the mechanism of LTA recognition critically differs from that of LPS. Starting from our first observation that activation of macrophages by LTA is significantly enhanced under serum-free conditions (Figs. 1 and 2), we investigated the role of serum and the serum protein LBP in cell activation by LTA. Employing biophysical techniques, we show that LTA interacts with soluble LBP, but it does not appear to interact with the membrane-associated form of LBP, nor is it transported into phospholipid membranes (Figs. 3 and 4).


Figure 9
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  		FIGURE 9.
Proposed model comparing LBP activation for LPS aggregates and inactivation for LTA with respect to intercalation into the phopsholipid matrix of the cytoplasmic membrane.

 
Using in vitro cell stimulation assays, we could identify LBP as an important protein in serum that attenuates cellular responses to LTA (Fig. 5). Also, the inhibitory effects of LBP are mediated by interaction of LTA with sLBP in serum, whereas the cell-associated form mLBP does not play a role in cell activation by LTA (Fig. 6). We have summarized our results of the mechanisms of cell activation by LTA in comparison with the mechanisms of cell activation by LPS (Fig. 9). This model depicts that the mechanisms of cell activation with respect to the role of LBP are quite different. Whereas the interaction of LPS with LBP is an important step enhancing the sensitivity of cell activation by mediating the transport of LPS to the cell surface and into the phospholipid matrix of the cytoplasmic membrane, the presence of LTA inhibits the intercalation of LBP into the membrane and other than LPS (17), LTA is not transported by LBP into the membrane. These results could be explained by the formation of LTA·LBP complexes leading to inactivation of LTA and LBP functions. A similar mechanism has been suggested for the neutralization of sLBP by phospholipids (39). Interaction of LTA with serum proteins has been reported previously, but the data published on the requirement of soluble LBP are conflicting. Thus, it has been reported that recombinant human LBP enhances the response of human monocytes to LTA preparations (32), whereas in another publication no influence of recombinant murine LBP on the cell activation of human monocytes could be observed (35, 36). The discrepancy of these observations might be due to species-specific effects or differences in the test systems employed. Because the requirement of CD14 in cell activation by LTA is not fully established, we also wanted to address this issue. Using blood-derived macrophages, we could show that under serum-free culture conditions cell activation by LTA was clearly inhibited in the presence of an anti-CD14 antibody. We wanted to confirm this result also for rat cells; however, the human anti-CD14 antibody does not cross-react with rat CD14. Thus, we made use of the action of PI-PLC, an enzyme known to cleave PI-anchored proteins such as CD14 from the cell surface (19, 47). PI-PLC treatment of primary rat alveolar macrophages led to a strong decrease of cellular response to LTA, confirming a role of mCD14 in cell activation by LTA. This is in accordance with the reports by Schröder et al. (32) showing a CD14 dependence of LTA-induced activation of human monocytes by inhibition with anti-CD14 antibodies and Lotz et al. (52), who showed a role for CD14 in the activation of neutrophil granulocytes by LTA. In contrast, Hermann et al. (36) described that cytokine production in human whole blood was inhibited by the addition of sCD14, and sCD14 was not able to confer a response to LTA on endothelial cells. This contradiction might reflect different roles of soluble and membrane-bound CD14 in cell activation by LTA. Very recently, a role for CD36 in cell activation by LTA has been shown (34). CD36 is a member of the scavenger receptor type B family and has been implicated to be involved in the recognition of oxidized LDL and fatty acids. The authors suggest that CD36 acts a facilitator for the recognition of LTA and functions analogously to CD14 in the LPS system.

An important aspect of our findings is the relevance for body compartments such as the lung that do not contain serum. The lung is in constant contact with microorganisms constantly inhaled by breathing. Each day, respiration moves about 13,000 liters of air containing microorganisms over the 200 m3 respiratory epithelial surface of the lung. Deposition of organisms induce complex strategies of the pulmonary innate immune system. Alveolar macrophages are a unique class of macrophages that function in innate immune defense of the lung against inhaled microorganisms. These cells are naturally adapted to a serum-free environment. Our observation that the response of alveolar macrophages to LTA is enhanced under serum-free culture conditions (Fig. 7) indicates that the pulmonary compartment exhibits a much more sensitive response to Gram-positive virulence factors than is exhibited in the circulation. Although LBP is predominantly produced in the liver and systemically circulated, recent studies indicate that LBP expression can also be induced by pulmonary type II epithelial cells (53). The concentrations of LBP in the lung greatly differ depending on the conditions. The concentration of LBP is assumed to be 10–100 ng/ml in the undiluted alveolar fluid, corresponding to a serum concentration of ~0.1–1% (54). In patients suffering from acute lung injury, bronchoalveolar concentrations of LBP have been documented to rise by ~100-fold (55). A recent paper reports that mice deficient in the expression of LBP have an impaired response to lung infection with Gram-negative organisms but not to pneumonia induced by Gram-positive organisms, supporting that LBP is not necessary for a sensitive innate immune response to Gram-positive-bacteria in the lung (56). Weber et al. (45) showed that the immune response to Gram-positive Streptococcus pneumoniae and pneumococcal cell wall preparations is facilitated by LBP and that meningeal inflammation upon pneumococcal infection are reduced in LBP-deficient mice. The authors identified the glycan backbone as the active component in cell wall preparations of S. pneumoniae that interacts with LBP. The participation and impact of Gram-positive virulence factors in immune activation and modulation in vivo will certainly have to be further elucidated in the future. In conclusion, our data indicate that LBP modulates the Gram-positive virulence factor LTA in a different mechanism from the Gram-negative virulence factor LPS, suggesting that compartmentalization is an important factor when studying innate immune reactions to infections on a molecular level.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants SCHR 621/2-1 and SCHR 621/2-2, SFB 367 Project B8, and STA 609/1-3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Research Center Borstel, Dept. of Immunochemistry and Biochemical Microbiology, Emmy-Noether Group Immunobiophysics, Parkallee 10, 23845 Borstel, Germany. Tel.: 49-4537-188296; Fax: 49-4537-188632; E-mail: aschromm{at}fz-borstel.de.

2 The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; PI-PLC, phosphatidylinositol-specific phospholipase C; PS, phosphatidylserine; TLR, toll-like receptor; TNF, tumor necrosis factor; sLBP, soluble LBP; mLBP, transmembrane configuration of LBP; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PE, phosphatidylethanolamine. Back


   ACKNOWLEDGMENTS
 
We thank C. Hamann, S. Groth, and S. Adam for excellent technical assistance and Prof. Dr. U. Zähringer for critically reading the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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