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Infection and Immunity, June 2003, p. 3663-3666, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3663-3666.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Stereochemistry of the Amino Acid Side Chain Influences the Inflammatory Potential of Muramyl Dipeptide in Experimental Meningitis

P. Cottagnoud,^1* C. M. Gerber,² P. A. Majcherczyk,³ F. Acosta,² M. Cottagnoud,² K. Neftel,² P. Moreillon,³ and M. G. Täuber⁴

Department of Internal Medicine, Inselspital,¹ Department of Internal Medicine, Zieglerspital,² Institute for Infectious Diseases, University of Bern, Bern,⁴ Department of Infectious Diseases, CHUV, Lausanne, Switzerland³

Received 25 July 2002/ Returned for modification 18 December 2002/ Accepted 19 March 2003

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Intrathecal injections of 50 to 100 µg of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF- secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF- secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF- release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

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Despite the availability of highly efficacious antibacterial treatment, bacterial meningitis remains one of the most serious infections. Streptococcus pneumoniae is the most common pathogen causing meningitis and is associated with mortality rates ranging between 19 and 27% and with neurological sequelae in approximately 30% of survivors (3, 8, 10). Recent discoveries in the pathophysiology of pneumococcal meningitis have contributed to a better understanding of the underlying mechanisms occurring during meningitis, highlighting the crucial role of the inflammatory response of the host (11). It has become clear that neurological sequelae are not due solely to the proliferation of bacteria in the subarachnoid space but are also the consequence of the excessive reaction of the immune system triggered by bacterial products. In pneumococci, the cell wall is the most potent proinflammatory stimulus which leads to activation of the cytokine cascade (14). The pneumococcal cell wall is a three-dimensional network of sugar chains with alternating N-acetylmuramic acid and N-acetyl-glucosamine, which are cross-linked by peptide side chains. Antibiotics causing bacterial lysis, e.g., ß-lactams, lead to a profound release of highly inflammatory cell wall pieces triggering the cytokine cascade in cerebrospinal fluid (CSF) (9, 12). The smallest inflammatory unit is believed to be a muramic acid with a peptide side chain (13).

The aim of this study was to investigate the role of the stereochemistry of the peptide side chain of the muramyl dipeptides with regard to their proinflammatory potential as determined by the influx of leukocytes and tumor necrosis factor alpha (TNF-) levels in the CSF of rabbits during experimental meningitis.

Rabbit meningitis model. The meningitis model, originally described by Dacey and Sande (2), was modified. The experimental protocol was accepted by the local ethical committee (Veterinäramt des Kantons Bern). Young New Zealand White rabbits (2 to 3 kg) were anesthetized by intramuscular injections of ketamine (30 mg/kg of body weight) and xylazine (15 mg/kg) and were immobilized in stereotactic frames. A lumbar punction needle was placed in the cisterna magna for CSF sampling. A long-acting anesthetic (ethyl carbamate [urethane]; 3.5 g/rabbit) was injected subcutaneously, and anesthesia was completed by repetitive intravenous injections of Nembutal. A total of 200 µl of CSF was removed (h 0) before muramyl dipeptides (MDPs) or heat-killed pneumococci were instilled intracisternally. Then, CSF was sampled at h 2, 4, 6, 8, and 10. Leukocyte (counted in a Neubauer chamber) and TNF- levels were determined. At the end of the experiment, euthanasia was induced by lethal intravenous doses of Nembutal. Seven rabbits were used in each group. As a control, 200 µl of NaCl was instilled intracisternally in three rabbits.

MDPs and heat-killed unencapsulated pneumococci. N-Acetylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-L-isoglutamine, and N-acetylmuramyl-D-alanyl-D-isoglutamine were commercially purchased (Sigma, St. Louis, Mo.). All MDPs have been synthesized with a purity rate of over 99%. They were diluted in sterile, pyrogen-free saline to final concentrations of 100 and 50 µg/200 µl and injected intracisternally at h 0.

S. pneumoniae R6, an unencapsulated pneumococcal strain (kindly provided by A. Tomasz, The Rockefeller University, New York, N.Y.), was grown in C+Y (5) to an optical density at 590 nm of 0.6 and then centrifuged, resuspended in saline, and boiled for 60 min. After boiling, cell viability was determined by plating an aliquot on blood agar plates. About 0.5 x 10⁸ heat-killed pneumococci in 200 µl of saline were injected intracisternally.

Preparation and stimulation of human peripheral blood mononuclear cells (PBMCs). The preparation of PBMCs was performed as described recently by Majcherczyk et al. (6). In brief, human PBMCs were isolated from blood of healthy volunteers by Ficoll-Hypaque density gradient centrifugation. Cells were resuspended in RPMI 1640 medium (Life Technologies, Inc.), and 0.5 x 10⁶/well were distributed into 96-well plates. Each well contained 140 µl of RPMI 1640 medium, 20 µl of plasma from the donor, and 20 µl of the sample to be tested. Lipopolysaccharide from Escherichia coli 0111 (Sigma Corporation) was used as a positive control in concentrations ranging from 0.01 to 100 ng/ml. The plates were incubated at 37°C in an atmosphere containing 5% CO₂. After 8 h of incubation, an aliquot of 20 µl was taken for TNF- measurement. Experiments were performed in triplicate.

Measurement of TNF-. TNF- levels in the CSF of rabbits and supernatants of PBMCs were determined as previously described (1, 6). In brief, using WEHI clone 13 murine fibroblast cells (10⁴/well), quantitation of TNF- was determined by measuring cytotoxicity. Recombinant murine TNF- was used as standard. The sensitivity of the assay was 25 pg/ml.

The proinflammatory potential of a series of MDP isomers was compared to that of entire cell walls from heat-killed unencapsulated pneumococci. Based on previous work by Tuomanen et al. (14), the amounts of intracisternally injected material were comparable, i.e., 10⁷ entire cells corresponded to approximately 20 µg of cell wall (MDP equivalents).

The effect of the presence of 50 µg of each of the three MDP isomers (L,D, L,L, and D,D) is shown in Fig. 1. At 2 h after intracisternal instillation, the L,D and L,L isomers had already induced pronounced TNF- secretion (between 4,000 and 14,000 pg/ml, with a peak ranging between 9,000 and 19,000 pg/ml 2 h later). Following the TNF- peak, leukocytes invaded the subarachnoid space, with numbers increasing progressively to a peak count of around 3,000 leukocytes/µl for both isomers at the end of the experimental period.

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FIG. 1. Effect of one intracisternal injection of 50 µg of either L,D MDP (squares) or L,L MDP (circles) on TNF- secretion and leukocyte influx into the CSF of rabbits. Filled symbols represent TNF- levels; empty symbols represent leukocytes.

Higher doses of the L,D and L,L MDPs (100 µg/rabbit) produced similar CSF inflammation, with the TNF- peak of 28,000 to 35,000 pg/ml occurring at only 2 h after injection. Leukocytosis levels were similar for both isomers (around 3,500 and 4,000 cells/µl after 10 h) (Fig. 2). In marked contrast, even at the higher dose (100 µg/rabbit; Fig. 3), the D,D isomer was completely inactive with regard to TNF- secretion and leukocytosis; during the entire treatment period, no significant TNF- secretion and CSF leukocytosis were detected. Figure 4 shows the effects of the presence of 0.5 x 10⁸ heat-killed unencapsulated pneumococci, corresponding to 100 µg of entire cell wall (or MDP). The progressive influx of leukocytes into the CSF was comparable to that induced by the corresponding dose of L,L or L,D MDP (2,400 ± 1,200 versus 3,000 ± 1,800 cells/µl for 50 µg of L,D MDP). However, leukocytosis was preceded by only negligible TNF- secretion. At 2 h after injection of the MDP, the TNF- level peaked at around 600 ± 202 pg/ml. During the entire experimental period, no TNF- secretion or leukocytosis was detected in the control (NaCl) group.

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FIG. 2. Effect of one intracisternal injection of 100 µg of either L,D MDP (squares) or L,L MDP (circles) on TNF- secretion and leukocyte influx into the CSF of rabbits. Filled symbols represent TNF- levels; empty symbols represent leukocytes.

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FIG. 3. Effect of one intracisternal injection of 100 µg of D,D MDP (squares) on TNF- secretion and leukocyte influx into the CSF of rabbits. Filled symbols () represent TNF- levels; empty symbols () represent leukocytes.

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FIG. 4. Effect of the presence of 107 heat-killed unencapsulated pneumococci on TNF- secretion () and leukocyte influx into the CSF space ().

Using cultures of human PBMCs, the potential of MDP isomers for induction of TNF- secretion was also tested in vitro. PBMCs were incubated with different concentrations of MDP isomers which corresponded closely to the amounts used in the rabbits. Instead of heat-killed pneumococci, purified cell walls were used. Cell walls were prepared as described previously (4). As a positive control, PBMCs were incubated with different lipopolysaccharide concentrations (0.1 to 100 ng/ml), leading to dose-dependent TNF- secretion (from 0 to 968 pg/ml; data not shown). The presence of L,L or L,D isomer led to a release of small amounts of TNF- from PBMCs (ranging between 100 and 200 pg/ml), without a clear dose-dependent effect (Fig. 5A and B). In analogy to results obtained in experimental meningitis, the D,D isomer was inert in this in vitro model (Fig. 5C). The most active compound was the complete pneumococcal cell wall, which caused dose-dependent TNF- secretion from PBMCs (Fig. 5D). The highest MDP concentration tested led to TNF- concentrations above 5,000 pg/ml.

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FIG. 5. Effect of different MDP isomers in different concentrations on TNF- secretion in human PBMCs after 8 h of incubation. (A) L,D isomer; (B) L,L isomer; (C) D,D isomer. (D) Dose-dependent TNF- secretion from human PBMCs after 8 h of incubation with different concentrations of whole pneumococcal cell walls (WCW) isolated from an unencapsulated strain.

Over the last decades, it has become clear that an ideal treatment for pneumococcal meningitis should include a highly bactericidal therapy combined with a potent adjunctive treatment counteracting the excessive reaction of the immune system responsible for neurological damage caused by the disease. It has also become evident that the deleterious cytokine cascade and leukocyte influx into the subarachnoid space is triggered by bacterial products. Thanks to efforts by several laboratories, it has been demonstrated that the major proinflammatory stimuli in pneumococcal meningitis are the cell wall and its components (teichoic acid and lipoteichoic acid) (14). Furthermore, increased release of these pneumococcal cell wall components occurs during cell wall autolysis due to endogenous autolysins (amidase), which are triggered in the late stationary phase of pneumococcal growth and by ß-lactam antibiotics.

The smallest proinflammatory units were obtained by further enzymatic degradation (by muramidase) of the cell wall to disaccharide peptides. The most proinflammatory disaccharide peptides carried an additional dipeptide substituent (alanyl-alanine or seryl-alanine) on the amino group of stem peptide lysine (isolated from the cell wall of a penicillin-resistant strain). In this experimental setting, isolated stem peptides after amidase digestion showed no proinflammatory activity (P. Cottagnoud, A. Severin, and A. Tomasz, 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 949, 1992).

The importance of small cell wall fragments as proinflammatory components resulting from cell wall autolysis was documented in previous work performed with the same rabbit meningitis model (A. Tomasz and P. Cottagnoud, 32nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 951, 1992). In that study, lysis-deficient pneumococci (Lyt^- mutants) carrying a point mutation in the major autolysin gene (LytA) produced less CSF leukocytosis (2 x 10³/µl) after a single injection of ampicillin than the wild-type organism (8 x 10³ to 10 x 10³/µl). The leukocytosis produced by the lysis-deficient strain might have been due partially to release of intracellular cell wall precursors not yet integrated into the intact cell wall, i.e., disaccharide pentapeptides. This would be in contrast to results for fragments in wild-type organisms generated from cell wall by autolysins.

The most important finding in the present study was to demonstrate the crucial role of the stereochemistry of the first amino acid (L-alanine) of the peptide side chain of MDPs. Whereas the L,L and L,D isomers produced dose-dependent TNF- secretion preceding the influx of leukocytes, the D,D isomer showed no activity. The importance of the stereochemistry of the first amino acid was also confirmed in vitro using cultures of PBMCs. Of interest, results with similar amounts of MDPs tested in both experimental settings showed that TNF- secretion was more pronounced in the animal model than in the cell culture system. Thus, while the two systems showed concordant results, the animal model was more sensitive to the effect of the proinflammatory bacterial products.

It is interesting that heat-killed unencapsulated pneumococci triggered the influx of leukocytes into the CSF with a negligible amount of preceding TNF- secretion, suggesting that the smallest cell wall unit and the whole cell wall produced different proinflammatory patterns. Furthermore, the whole cell wall was the most active compound in inducing TNF- secretion in vitro, whereas the amount of TNF- in CSF produced by intracisternally injected heat-killed pneumococci was negligible. Since it is assumed that the pneumococcal cell wall presented in heat-killed unencapsulated organisms is stereochemically similar to that present in cell wall preparations, the latter observation may point to a significant difference in inflammatory pathways between the rabbit model and the human white blood cell system. A recently published study of Chinese hamster ovary fibroblasts by Yoshimura et al. (16) confirmed the diversity of inflammatory pathways triggered by cell wall components. These authors demonstrated that after hydrolysis by N-acetylmuramidase, the cell wall of Staphylococcus epidermidis was not active at all, contrasting with the results for pneumococcal cell wall hydrolysate obtained with our rabbit model (after digestion with muramidase; see above).

These observations raise a question regarding the exact mechanisms by which components of the pneumococcal cell wall signal to host cells to induce an inflammatory response. It is generally assumed that peptidoglycans of gram-positive organisms signal through Toll-like receptor 2-dependent pathways (7, 15). Our results indicate that (i) the signaling capacity of MDPs depends on the stereochemistry of the first amino acids of the dipeptide and that (ii) the signaling is different for small glycopeptides compared to that of undigested cell walls present in heat-killed, unencapsulated pneumococci. The basis for these observations is currently unclear, but it is conceivable that signaling occurs through receptors other than Toll-like receptor 2. The search for additional receptors through which components of the pneumococcal cell wall may be able to induce a proinflammatory signal may provide new insights into the complex interactions between the pneumococcal pathogen and its mammalian host.

 
* Corresponding author. Mailing address: Department of Internal Medicine, Inselspital, Freiburgstrasse, 3010 Bern, Switzerland. Phone: 41-31-632-34-72. Fax: 41-31-632-38-47. E-mail: pcottagn{at}insel.ch.

Editor: V. J. DiRita

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Infection and Immunity, June 2003, p. 3663-3666, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3663-3666.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cottagnoud, P. Articles by Täuber, M. G. Search for Related Content PubMed PubMed Citation Articles by Cottagnoud, P. Articles by Täuber, M. G.