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Definition of Structural Prerequisites for Lipoteichoic Acid-Inducible Cytokine Induction by Synthetic Derivatives¹

Susanne Deininger^*, Andreas Stadelmaier, Sonja von Aulock^*, Siegfried Morath^*, Richard R. Schmidt and Thomas Hartung^2,*

^* Biochemical Pharmacology and Department of Chemistry, University of Konstanz, Konstanz, Germany

	Abstract

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The controversy about the immune stimulatory properties of lipoteichoic acid (LTA) from Staphylococcus aureus was solved recently by showing decomposition and inactivation of LTA obtained by conventional purification strategies, as well as pronounced LPS contamination of commercial preparations. By introducing a novel preparation method, the structure of bioactive LTA was elucidated. This structure was confirmed by chemical synthesis. In this work, synthetic LTA derivatives were employed to study the structure-function relationship of cytokine induction in human monocytes. Synthetic LTA induced the same cytokine pattern as highly purified natural LTA. The gentiobiose core could be omitted without affecting bioactivity. The polyglycerophosphate backbone amplified the response to the lipid anchor (100-fold) only when substituted with D-alanine, whereas -D-N-acetylglucosamine substituents could be omitted. Replacing D-alanine substituents with L-alanine reduced the activity of the molecule at least 10-fold, indicating stereoselectivity. These results define for the first time the crucial patterns required for the immune recognition of LTA.

	Introduction

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Because we carry ten times more commensal bacteria than human cells, the boundaries to the inner compartments of the body must be protected carefully by the immune system. However, in case of a bacterial infection that cannot be controlled, the activation of the immune system may escalate to a life-threatening sepsis. For Gram-negative bacteria, LPS is well established as the crucial stimulus of the innate immune system, as injection into mice causes all known symptoms of sepsis (1). There is increasing evidence that lipoteichoic acid (LTA)³ from the cytoplasmic membrane of Gram-positive bacteria is an immunostimulatory Gram-positive counterpart to LPS (2). The importance of Gram-positive organisms in hospital infections has increased in the past three decades. In the last decade, Gram-positive organisms were identified as frequently as Gram-negative organisms as the cause of sepsis (3). Staphylococcus aureus is the most frequently isolated Gram-positive pathogen that causes infections (4), e.g., in trauma patients (5, 6). A further problem, apart from the increase of S. aureus infections, is the development of antibiotic resistance in this species (7, 8). Many S. aureus strains are increasingly reported to resist all current antibiotics except for vancomycin, and now even strains with additional vancomycin resistance are found (9). Therefore, alternatives and adjuvants to antibiotics are requested. To identify these, it is important to understand the pathophysiology of this bacterial infection. Thus, we have addressed the role of lipoteichoic acid, which appears to represent a key pathogen-associated molecular pattern of S. aureus in immune activation.

LTA molecules consist of up to 50 repeating poly-(polyolphosphate) units fixed in the cytoplasmatic membrane by a lipid anchor. They appear to be crucial for the vital functions of the bacteria because their biosynthesis is not halted even in case of phosphate deficiency. Their biological functions identified so far include binding of metal cations by the negatively charged polysaccharide chains (charge densities are regulated by amino acid substituents like D-alanine and D-lysine) (10) and regulation of the activity of autolytic enzymes (11, 12). To the host, they represent patterns for immune recognition, similar to LPS.

The amphiphilic LTA molecule resembles LPS in that it also forms micelles, is also lipid anchored, and carries phosphate and repetitive subunits of carbohydrates. It is known that LTA is recognized by macrophages via membrane-associated CD14 and Toll-like receptor (TLR) 2, but does not require participation of LPS-binding protein in humans (13, 14). Published results show that commercial LTA preparations are often of poor quality. In many cases, significant activities reported could be attributed to LPS contaminations (15, 16). Furthermore, appropriately purified, phenol-extracted LTA from S. aureus was found to be unable to induce cytokine release, a measure of immunostimulatory activity (17). We have recently attributed this to a decomposition of LTA resulting during the usual phenol extraction procedure (18). This decomposition was characterized by a loss of glycerophosphate units as well as D-alanine and -D-N-acetylglucosamine (GlcNAc) substituents from the LTA backbone. Meanwhile, it is possible to purify essentially homogenous, biologically active LTA by a butanol extraction method described recently (19). Furthermore, the chemical synthesis of biologically active LTA based on the structure of LTA from S. aureus was conducted (20). Using this synthetic LTA, we confirmed the key role of D-alanine substituents for the immunostimulatory activity by exchanging them with L-alanine. This derivative was less active in the stimulation of TNF- release from whole blood than was the compound with D-alanine. Furthermore, we observed that the lipid anchor alone, after separation from the backbone or when synthesized chemically, still retained immunostimulatory potential. However, the concentration-response curve was shifted to higher concentrations by at least two log-orders. After loss of one of the two fatty acids, the LTA anchor lost its cytokine stimulating activity (20). Here we wanted to extend these results to define the structure-function relationship of the LTA molecule and relate it to cytokine patterns. For this purpose we used chemically synthesized and artificially modified LTA derivatives lacking defined subunits.

	Materials and Methods

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Stimuli

The general strategy of synthesis of lipoteichoic derivates was adopted with modifications as reported (20). The detailed synthesis has been published (22). The identities of the molecules were characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and nuclear magnetic resonance (NMR) at 600.13 MHz and 300 K. The NMR spectra were related to 3-(trimethysilyl) 3,3,2,2-tetradeuteropropionic acid Na salt (d₄-TSPA). According to NMR data, purities of LTA derivates were >98%. The synthetic LTA and the biological LTA were negative in the Limulus test for Gram-negative endotoxin (i.e., <1 IU LPS/mg LTA, Endosafe Limulus amoebocyte lysate Endochrome; Charles River Laboratories, Charleston, SC), excluding significant LPS contamination. The m.w. of the substances was 2248.90 for LTA with D-alanine (D-Ala-LTA) or with L-alanine (L-Ala-LTA), 1924.70 for LTA without gentiobiose, and 2116.8 for LTA in which GlcNAc was replaced with a D-alanine. LPS from Salmonella abortus equi was purchased from Sigma-Aldrich (Deisendorfen, Germany).

Whole blood cytokine response

Differential blood cell counts were routinely performed with a Pentra 60 (ABX Diagnostics, Montpellier, France) to exclude acute infections of blood donors. Incubations of human whole blood with LTA derivatives were performed essentially as described (21). Briefly, 200 µl heparinized blood freshly taken from healthy volunteers was diluted 5-fold with isotonic sodium chloride solution (0.9%; Boehringer Ingelheim, Ingelheim, Germany). LTA was sonified before use to dissolve micelles. After addition of the LTA stimuli in a volume of 10 µl, incubations were conducted in polypropylene reaction tubes (Brinkmann Instruments, Hamburg, Germany) in the presence of 5% CO₂ at 37°C overnight. Cell-free supernatants were obtained by centrifugation at 400 x g for 2 min and stored at -70°C for cytokine determination.

Animals

TLR2 knockout mice were generated by homologous recombination and kindly provided by Tularik (South San Francisco, CA). TLR2 receptor-deficient mice and the corresponding wild-type mice (129Sv/B57BL/6) were bred in the animal house of the University of Konstanz (Konstanz, Germany). Husbandry conditions were 25°C, humidity 55%, 12-h day-night rhythm, and nutrition with Altromin C 1310 (Altromin, Lage, Germany). All animals received humane care in accordance with the National Institutes of Health guidelines and the legal requirements in Germany.

Generation of peritoneal cell populations

Mice were put to sleep by terminal pentobarbital anesthesia (Narcoren; Merial, Halbergmoos, Germany) by i.v. injection into the tail vein. Ice-cold PBS (10 ml; PAA Laboratories, Linz, Austria) was injected into the peritoneal cavity after skinning the abdomen. Mice were shaken gently, and the lavage liquid was transferred into siliconized glass tubes (Vacutainer; BD Biosciences, Plymouth, U.K.) and centrifuged at 300 x g for 10 min. The cell pellet was resuspended in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) containing 10% FCS (heat-inactivated; Biochrom, Berlin, Germany) and 100 IU/ml penicillin/streptomycin (Biochrom).

Incubation conditions of peritoneal cell populations

Cell populations were transferred to 96-well cell culture plates (Cellstar; Greiner, Frickenhausen, Germany) at 10⁵ peritoneal cells/well. Each well contained a total volume of 200 µl RPMI 1640 (10% FCS and 100 IU/ml penicillin/streptomycin). For the determination of cytokine induction by LTA or LPS, cells were incubated with stimuli dissolved in pyrogen-free saline (Ampuwa; Fresenius Medical Care, Bad Homburg, Germany) at 37°C, 5% CO₂, and humidified air overnight. Cell-free supernatants were stored at -70°C.

Cytokine ELISAs

Cytokines were measured by sandwich ELISA based on Ab pairs against human IL-1, IL-8, TNF- (Endogen, Eching, Germany), IL-10 (BD PharMingen, Hamburg, Germany), and IL-6 (R&D Systems, Wiesbaden, Germany) and against murine IL-6 (BD PharMingen). Recombinant cytokines serving as standards were a gift from Dr. S. Poole, National Institute for Biological Standards and Controls (London, U.K.) (TNF-, IL-1), or purchased from BD Biosciences (IL-10), PeproTech/TEBU (Frankfurt, Germany) (IL-8), Genzyme (Ruesselsheim, Germany) (IL-6), or BD PharMingen (murine IL-6). Binding of biotinylated Ab was quantified using streptavidin-peroxidase (BioSource International, Camarillo, CA) and the substrate TMB (3,3',5,5'-tetramethylbenzidine; Sigma-Aldrich).

Statistics

Data are presented as means ±SEM. Statistical significance was determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test (data were normalized by a log transformation). In case of two groups, data were analyzed by one-tailed paired t test, which are both options of the GraphPad InStat 3.00 software (GraphPad, San Diego, CA). Cytokine levels are given per milliliter of blood, i.e., corrected for a dilution factor of 5 in the 20% blood incubation.

	Results and Discussion

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Synthetic LTA induces a cytokine pattern similar to native LTA

We recently synthesized a complete LTA molecule based on the structure of S. aureus LTA. It consists of a gentiobiosyl-sn-dimyristoylglycerol anchor joined to six glycerophosphate units carrying four D-alanine and one GlcNAc substituent and is therefore shorter than native LTA, which has 30–50 glycerophosphate units (19). Comparison of the release of several cytokines in human whole blood showed that synthetic and native LTA have the same potency and pattern (Fig. 1), except for some TNF- low responders who produced <10 pg TNF-/ml in response to the synthetic LTA. This implies that the synthetic LTA reflects the key structural requirements for immune activation as found in native LTA.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Cytokine pattern induced by synthetic and native LTA. Comparison of cytokine release by 1 µg/ml synthetic LTA and equimolar 4.8 µg/ml native LTA in human whole blood. Values are for six donors. TNF-, IL-8, IL-6, IL-1, and IL-10 levels are given as nanograms per milliliter of blood.

We confirmed our previous results concerning TNF--release (20) and extended the assessment of immunostimulatory activity of the LTA derivatives D-Ala-LTA and L-Ala-LTA and the LTA anchor by measurement of IL-1, IL-6, IL-10, and IL-8 (structures shown in Fig. 2, A and B).

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Chemical structure of synthetic LTA and LTA anchor. Structure of synthetic LTA (D-Ala-LTA) with six glycerophosphate units carrying four alanine and one N-acetylglucosamine substituent, based on the LTA structure for S. aureus (A) and LTA anchor and based on the LTA structure for S. aureus (B).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Comparison of LTA with D-alanine, LTA with L-alanine, and LTA anchor-induced IL-8 release. Concentration dependence of IL-8 response of human whole blood to D-Ala-LTA, L-Ala-LTA, and LTA anchor. Data are means ±SEM of twelve donors. Significant difference (***, p < 0.001 vs control (NaCl); #, p < 0.05; ###, p < 0.001 vs D-Ala-LTA) based on one-way ANOVA, followed by Dunnett‘s Multiple Comparison Test.

Replacing D-alanine by L-alanine attenuated the stimulatory activity indicating stereoselective recognition. It might be speculated that D-alanine substituents enable ionic interactions between LTA molecules forming multimers. Either L-alanine does not enable ionic interactions as well as D-alanine, or L-Ala-LTA itself is not recognized equally well by monocyte receptors.

Based on these results which indicate that the LTA anchor alone has little immunostimulatory activity, we utilized further modified LTA molecules to characterize functional requirements for LTA activity. Next, two molecules were synthesized which lack either the GlcNAc or the gentiobiose subunits.

Role of GlcNAc substituents in the induction of cytokine release by LTA

First we used the synthetic LTA that corresponds to D-Ala-LTA without GlcNAc substituents. GlcNAc was replaced by a further D-alanine substituent. We tested whether the absence of GlcNAc had an influence on cytokine release in comparison to D-Ala-LTA. The cytokine release was of a similar extent in response to D-Ala-LTA and synthetic LTA without GlcNAc over a concentration range from 1 ng/ml to 10 µg/ml regarding TNF-, IL-1, and IL-8 (Fig. 4) as well as IL-6 and IL-10 (not shown). Both induced a whole blood cytokine response starting at as little as 100 ng/ml and increasing steadily up to 10 µg/ml. It seems that the GlcNAc substituent plays no decisive role in the biological activity of LTA.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Cytokine stimulatory activity of D-Ala-LTA compared with LTA without GlcNAc (replaced by D-alanine). Human whole blood was incubated overnight with either derivative. D-Ala-LTA is m.w. 2248.90 and LTA without GlcNAc is m.w. 2116.80. Cytokines were determined in cell-free supernatants by ELISA. Data are means of seven donors ±SEM.

Cytokine response to D-Ala-LTA and LTA without gentiobiose

Next, we constructed a synthetic LTA that does not possess the gentiobiose subunit. We again quantified the release of several cytokines by stimulation with concentrations from 1 ng/ml to 10 µg/ml (Fig. 5). The loss of gentiobiose also had no significant effect on cytokine release. All cytokine levels measured were similar for D-Ala-LTA and LTA without gentiobiose. Therefore, we conclude that the gentiobiose does not represent an important factor for the immunostimulatory activity of LTA.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Cytokine induction of D-Ala-LTA compared with LTA without gentiobiose. Human whole blood was stimulated overnight with either D-Ala-LTA (m.w. 2248.90) or LTA without gentiobiose (m.w. 1924.70) as indicated. Cytokines were determined in the cell-free supernatants by ELISA. Data are means of four to eight donors ±SEM.

Peritoneal cell populations from TLR2 knockout mice and wild-type mice (n = 4) were stimulated with 10 µg/ml of native LTA, D-Ala-LTA, L-Ala-LTA, LTA without gentiobiose, LTA without GlcNAc or endotoxin (LPS). After overnight incubation, murine IL-6 was measured in the supernatants by sandwich ELISA. IL-6 release after stimulation with native LTA or LTA derivatives of TLR2-deficient peritoneal cells did not surpass control levels (0.11 ± 0.03 ng IL-6/ml), whereas native LTA (11.9 ± 1.5 ng IL-6/ml) and all LTA derivatives (e.g., D-Ala-LTA, 4.4 ± 0.7 ng IL-6/ml; LTA without gentiobiose, 2.5 ± 0.9 ng IL-6/ml) induced IL-6 in wild-type cells. We conclude that TLR2 is the receptor employed by native LTA as well as LTA derivatives. IL-6 release in response to stimulation with LPS was the same in TLR2-deficient and wild-type cells (7.3 ± 0.6 vs 8.2 ± 0.9 ng IL-6/ml, respectively).

Taken together, the key components of the LTA molecule necessary for stimulation of monocytes are the LTA anchor with two fatty acids and a glycerophosphate backbone with D-alanine substituents. The exchange of D-alanine by L-alanine reduced the immunostimulatory potency significantly, which implies stereoselective recognition. In addition, we observed that the loss of the GlcNAc or gentiobiose had no decisive effect on the cytokine release in human whole blood, indicating that these components are not required for host recognition. It remains to be elucidated which minimal structure of LTA is required for potent cytokine release, i.e., we next intend to examine the immunostimulatory potential of LTA with a minimal backbone.

	Acknowledgments

 
We are grateful for the excellent technical assistance by L. Cobianchi, J. Kley, G. Pinski, S. Reichstein, I. Seuffert and M. Kreuer-Ullmann, and for the help of S. Hoffmann with the statistical analysis. We express our gratitude to Prof. A. Geyer for supervising structural analysis by NMR spectroscopy.

	Footnotes

 
¹ This work was supported by Deutsche Forschungsgemeinschaft (Grant For 434/1-1, TP 6) and the Fonds der Chemischen Industrie.

² Address correspondence and reprint requests to Dr. Thomas Hartung, Biochemical Pharmacology, University of Konstanz, 78457 Konstanz, Germany. E-mail address: Thomas.Hartung{at}uni-konstanz.de

³ Abbreviations used in this paper: LTA, lipoteichoic acid; L-Ala-LTA, LTA with L-alanine; D-Ala-LTA, LTA with D-alanine; GlcNAc, N-acetylglucosamine; NMR, nuclear magnetic resonance; TLR, Toll-like receptor.

Received for publication September 23, 2002. Accepted for publication January 24, 2003.

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