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The Journal of Immunology, 2006, 177: 1221-1228.
Copyright © 2006 by The American Association of Immunologists

Streptococcal M Protein: A Multipotent and Powerful Inducer of Inflammation1

Lisa I. Påhlman*, Matthias Mörgelin*, Jana Eckert{dagger}, Linda Johansson{ddagger}, Wayne Russell2,*, Kristian Riesbeck§, Oliver Soehnlein, Lennart Lindbom, Anna Norrby-Teglund{ddagger}, Ralf R. Schumann{dagger}, Lars Björck* and Heiko Herwald3,*

* Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Lund University, Lund, Sweden; {dagger} Institute for Microbiology and Hygiene, Charité University Medical Center, Humboldt University, Berlin, Germany; {ddagger} Karolinska Institutet, Center for Infectious Medicine, Huddinge University Hospital, Stockholm, Sweden; § Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, Malmö, Sweden; and Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Severe infections with Streptococcus pyogenes, an important human pathogen, are associated with massive inflammatory reactions in the human host. Here we show that streptococcal M protein interacts with TLR2 on human peripheral blood monocytes. As a consequence, monocytes express the cytokines IL-6, IL-1beta, and TNF-{alpha}. This response is significantly increased in the presence of neutrophil-derived heparin-binding protein (HBP), which costimulates monocytes by interacting with CD11/CD18. Analysis of tissue biopsies from patients with necrotizing fasciitis revealed recruitment of neutrophils and monocytes to the infectious site, combined with the release of HBP. The results show that M protein, in synergy with HBP, evokes an inflammatory response that may contribute to the profound pathophysiological consequences seen in severe streptococcal infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune response constitutes the first line of defense against invading pathogens. To achieve a broad spectrum of action, the early response is dependent on nonclonal recognition of microbial components (1). Thus, cells that are involved at this stage of the host response, primarily polymorphonuclear neutrophils (PMNs),4 peripheral blood monocytes, and dendritic cells, express so-called pattern recognition receptors (PRR) that bind to and eliminate bacteria or bacterial products. Upon binding, these compounds, also referred to as pathogen-associated molecular patterns, are able to trigger an inflammatory response by activating various signaling pathways (2). The best known PRRs are the TLRs (3), but the list of PRRs is growing and consists of other receptors such as NOD proteins as well as complement and scavenger receptors (4, 5). TLRs belong to the family of type I transmembrane receptors, with an extracellular leucine-rich repeat region and a cytoplasmic domain (Toll/IL-1 receptor or TIR domain) that shares homology with the IL-1 receptor (6). Signaling occurs via a MyD88-dependent or MyD88-independent pathway that eventually leads to an activation of transcription factors such as NF-{kappa}B (7, 8). As a consequence, the cells express genes encoding for instance proinflammatory cytokines, that are required for an effective host response. Hitherto, 10 TLRs have been described in humans (8) and, with the exception of TLR10, at least one ligand has been identified for each receptor. As expected, most of the ligands are of bacterial or viral origin, including LPS, flagellin, unmethylated CpG DNA of bacteria and viruses, and dsRNA (9, 10), but there are also reports that host-derived proteins such as heat shock proteins, are capable of acting as endogenous activators of TLRs (11).

In severe infectious diseases, the concentrations of pro- and anti-inflammatory cytokines often reach pathological levels. The highest values are found in plasma samples of nonsurviving patients (12). In invasive Streptococcus pyogenes infections, one of the most feared conditions in infectious medicine, patients exhibit disturbed microcirculation, hypotension, shock, and organ failure with mortality rates that can exceed 50% (13, 14). Recently, we reported a novel virulence mechanism used by S. pyogenes to evoke vascular leakage by triggering human PMNs to release heparin-binding protein (HBP) upon stimulation with M protein released from the bacterial surface (15). M proteins are classical virulence determinants of S. pyogenes, that in 1969 were already shown to promote survival of the bacterium in human blood (16). The proteins are {alpha}-helical coiled coil surface proteins which are normally attached to the cell wall of S. pyogenes bacteria (17) but can be released spontaneously or by the action of endogenous or host proteinases (15, 18, 19).

In the present work, we extend our studies on molecular mechanisms used by M protein to induce inflammatory responses. The results show that M protein is capable of stimulating monocytes via TLR2 to produce high amounts of proinflammatory cytokines and that this effect is further augmented by HBP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

M1 protein was purified as described from the isogenic mutant MC25 strain (derived from the AP1 strain; 40/58 S. pyogenes strain from the World Health Organization Collaborating Centre for references and Research on Streptococci, Institute of Hygiene and Epidemiology, Prague, Czech Republic) that lacks the membrane-spanning region and secretes a soluble form of the protein (20). Briefly, the supernatant from an MC25 culture was collected, proteins were precipitated with ammonium sulfate, dissolved in PBS, and purified on fibrinogen-coupled Sepharose. The purity of the M1 protein preparation was confirmed by SDS-PAGE analysis followed by Coomassie or silver staining. The peptidoglycan and lipoteichoic acid contents in the stock solution of M1 protein (0,5 mg/ml) were below detection limit (<100 ng/ml for peptidoglycans and <3 ng/ml for lipoteichoic acid) as analyzed by mass spectroscopy and ELISA. M3 protein, M5 protein, M49 protein, protein H, IdeS, protein FOG, protein PAB, protein SIC, the streptococcal cysteine proteinase SpeB, and recombinant human HBP were purified as previously described (18, 19, 21, 22, 23, 24, 25, 26). HBP was purified according to good manufacturing practice guidelines and was free of endo- and exotoxins. Protein G was obtained from Amersham Biosciences, protein A and LPS were from Sigma-Aldrich, anti-TLR2 was from Santa Cruz Biotechnology, anti-HBP was from Leukotech, and the control Ab, unspecific rabbit IgG, was from DAKO. Plasmids encoding beta-galactosidase and the ELAM NF-{kappa}B reporter plasmid were kindly provided by C. J. Kirschning (University of Munich, Munich, Germany). All other reagents were purchased from Sigma-Aldrich unless indicated otherwise.

Purification of PBMCs

Human PBMCs were prepared from fresh heparinized blood from healthy volunteers. The blood was diluted 1/1 in PBS (Invitrogen Life Technologies) and layered on top of Ficoll-Paque Plus (Amersham Biosciences). After centrifugation at 1000 rpm for 20 min at room temperature, the PBMC layer was removed and washed twice in PBS or medium.

Cell lines and culture conditions

Mono Mac 6 (MM6) cells, a monocytic cell line (27), were maintained in RPMI 1640 supplemented with 10% (v/v) FCS, streptomycin at 100 µg/ml, penicillin at 100 U/ml, 1 mM sodium pyruvate, 1x MEM nonessential amino acid solution (Sigma-Aldrich), 1 mM oxaloacetic acid, and human insulin at 10 µg/ml. Cultures were seeded at a density of 2 x 105 cells/ml. Cell culture medium, PBS, and antibiotics were obtained from Invitrogen Life Technologies, whereas supplements for MM6 medium were purchased from Sigma-Aldrich.

Human embryonic kidney cells (HEK) 293 were maintained in DMEM (Invitrogen Life Technologies) including sodium pyruvate and supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 200 mM L-glutamine. All cell culture experiments were conducted under sterile conditions, and cells were cultured at 37°C in 5% CO2-95% air humidified incubator.

Cytokine detection by ELISA

Heparinized blood (diluted 1/10 in PBS) or freshly prepared PBMCs (2.5 x 106 cells/ml in PBS) were stimulated with bacterial products for 24 h at 37°C on rotation. Cells were pelleted by centrifugation, and the concentrations of IL-6, IL-1beta, and TNF-{alpha} in the supernatants were determined by ELISA according to the manufacturer’s protocol (R&D Systems).

Analysis of intracellular cytokines by flow cytometry

Human PBMCs were adjusted to 2.5 x 106 cells/ml in RPMI 1640 supplemented with 10% (v/v) FCS, 50 µg/ml gentamicin, 1 mM sodium pyruvate, 10 mM HEPES, and 50 µM mercaptoethanol (Invitrogen Life Technologies). Cells were stimulated with M1 protein (1 µg/ml, final concentration) with or without HBP (10 µg/ml, final concentration) in the presence of brefeldin A (3 µg/ml, final concentration) and incubated for 24 h at 37°C. The monocyte population was identified by staining with anti-CD14-RPE (DAKO) before fixation in 2% (v/v) paraformaldehyde in PBS for 10 min. Samples were permeabilized in 0.5% (w/v) saponin, 1% (w/v) BSA, and 0.1% (v/v) NaN3 in PBS for 20 min on ice and then stained with anti-IL-6-FITC, anti-IL-1beta-FITC, or anti-TNF-anti-FITC (R&D Systems). As a control, samples were incubated with 30x molar excess of IL-6, TNF-{alpha} (AL-ImmunoTools), or IL-1beta (BD Biosciences), which completely abolished the binding of the corresponding Ab to intracellularly stored cytokines. Samples were analyzed in a FACSCalibur flow cytometer. The monocyte population was identified by forward scatter (FSC)/side scatter (SSC) characteristics and CD14 expression.

Binding assays

M1 protein and HBP were FITC conjugated with EZ-Label FITC Protein Labeling Kit (PIERCE Biotechnology) according to the instructions provided by the manufacturer. Heparinized blood was washed twice in PBS and incubated with FITC-labeled M1 protein (1 µg/ml, final concentration) or HBP (10 µg/ml, final concentration) on rotation for 1 h at 37°C. After two washing steps, 100 µl of sample were fixed in 250 µl of Fix buffer (9.25% (v/v) formaldehyde and 3.25% (v/v) methanol) for 60 s. Erythrocytes were subsequently lysed in 4 ml of Tyrode’s buffer (137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM Na2HPO4, 0.35% (w/v) BSA, 10 mM HEPES, 5.5 mM dextrose, pH 7.4) for 20 min at room temperature. Alternatively, freshly prepared PBMCs (2.5 x 106 cells/ml) were incubated with FITC-labeled M1 protein (1 µg/ml) or HBP (10 µg/ml) for 1 h at 37°C. The samples were washed in PBS and analyzed in a FACSCalibur flow cytometer. Cells were gated based on FSC and SSC characteristics. Coincubation with 100x excess of unlabeled M1 protein or HBP-reduced binding of the FITC-labeled protein by 90 and 60%, respectively.

Stimulation of CHO cells and measurement of CD25 up-regulation

Transfected Chinese hamster ovarian (CHO) cells expressing CD14 and TLR2 or TLR4 (CHO/CD14/TLR2 and CHO/CD14/TLR4) (28, 29) were cultured in Ham’s F-12 medium supplemented with 10% (v/v) FCS and hygromycin B (400 U/ml). Geneticin (0.5 mg/ml) was used for TLR2-transfected cells, and puromycin was used (50 µg/ml) for TLR4-transfected cells. CHO cells were grown in 6-well plates until they were confluent and stimulated overnight at 37°C with M1 protein at different concentrations, protein PAB (1 µg/ml), Escherichia coli LPS (0.5 µg/ml; Sigma-Aldrich), or formaldehyde-treated Staphylococcus aureus. After trypsin treatment, detached cells were washed once in PBS containing 2% (w/v) BSA and incubated with RPE-conjugated anti-CD25 mAb (Dakopatts) for 30 min on ice. After a washing, samples were analyzed by flow cytometry. To ensure TLR expression, transfectants were stained with anti-TLR4 (clone HTA 125) and anti-TLR2 (clone TLR2.1), mAbs that were kindly provided by Dr. T. Espevik (Norwegian University of Science and Technology, Trondheim, Norway). Bound Abs were detected with a FITC-conjugated goat anti-mouse Ab and analyzed by flow cytometry.

Transient transfection of HEK 293 cells and measurement of luciferase activity

Transient transfection of HEK 293 cells was performed using FuGENE 6 (Roche Applied Science) transfection reagent following the manufacturer’s instruction. Cells (1 x 105 cells/well) were plated in 12-well plates and incubated overnight. At a density of 50–80%, they were transfected with plasmids containing 120 ng of NF-{kappa}B reporter luciferase and 40 ng of Rous sarcoma virus-beta-galactosidase (to correct for differences in transfection efficacy), and were cotransfected with human TLR2 (50 ng). Twenty-four hours after transfection, cells were stimulated with various potential ligands or controls diluted in DMEM containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 200 mM L-glutamine. Twenty hours after stimulation, cell extracts were prepared for determination of luciferase activity using luciferase assay reagents (Roche Applied Science) according to the manufacturer’s instruction. Luciferase and beta-galactosidase activity was estimated by a chemiluminescence-based assay (Boehringer).

Intracellular Ca2+ mobilization

MM6 cells were incubated (37°C for 30 min) with the Ca2+-sensitive fluorophore fluo-4/AM (Molecular Probes) according to the manufacturer’s instructions and washed twice with PBS before use. Ab IB4 (30) or an isotype control Ab (10 µg/ml) were added to the labeled MM6 suspension 30 min before injection of HBP. MM6 cells (500 µl; 106 cells/ml) were subjected to stimulation with HBP (500 ng/ml) or M1 protein (1 µg/ml). Changes in intracellular free Ca2+ were analyzed by flow cytometry (FACSort; BD Biosciences).

Thin sectioning and transmission electron microscopy

Abs against M1 protein and TLR2 were labeled with either citrate gold (15 nm in diameter) or SCN-gold (4 nm in diameter), respectively, as described (31). Human monocytes were incubated with M1 protein (1 µg/ml) for 30 min at 37°C. After a washing in PBS, cells were pelleted by centrifugation and subsequently fixed and sectioned. Thin sections were subjected to immunolabeling as described (32) with the modification that Aurion-BSA (Aurion) was used as a blocking agent. Samples were finally stained with uranyl acetate and lead citrate and observed in a Jeol JEM 1230 electron microscope, operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 charge-coupled device camera.

Analyses of HBP, PMNs, and monocytes/macrophages in patient biopsies

Snap-frozen biopsies of tissue (n = 8) collected from the epicenter of infection from patients with severe soft tissue infections caused by M1T1 S. pyogenes strains (tissue material kindly provided by Professor Donald E. Low, Mount Sinai Hospital, Toronto, Canada and Drs. Per Åkesson and Adam Linder, Lund University Hospital, Lund, Sweden) were assessed for the presence of bacteria, HBP release, PMNs and macrophage/monocyte cellular infiltrates. The study was performed in accordance with the Declaration of Helsinki, and ethical approval to obtain the biopsies was granted by the Human Subjects Review Committee of the University of Toronto and regional ethical committee in Lund, Sweden. The biopsies were cryosectioned, fixed, immunohistochemically stained, and analyzed by image analysis as previously described (33). The staining method was modified to include an initial blocking step with 20% (v/v) FCS in balanced salt solution-saponin for 30 min at room temperature followed by 1% (v/v) normal goat serum for 30 min. Monocytes/macrophages were identified by use of anti-CD68 (EBM11, murine IgG1; Dako), PMNs by anti-neutrophil elastase (NP57, murine IgG1; Dako), and HBP was identified by purified IgG from polyclonal anti-HBP rabbit antiserum (34). S. pyogenes organisms were identified by use of a polyclonal rabbit antiserum specific for the Lancefield group A carbohydrate (Difco). Despite the size of the biopsies, which varied significantly, the whole section was analyzed by acquired computerized image analysis (ACIA), yielding an analyzed cell area (defined by blue hematoxylin counterstain) ranging from 0.2 x 105 to 7.2 x 105 mm2. The results are presented as ACIA values, which equals the percentage of positively stained area times the mean intensity of positive staining. Sections stained in an identical manner but with exclusion of primary Abs served as controls for nonspecific staining and were completely negative for all biopsies.

Immunofluorescence staining of HBP and respective cell marker were performed and analyzed by confocal microscopy. The staining procedure has previously been described in detail (15). HBP was identified by use of streptavidin-conjugated Alexa Fluor 488 (Molecular Probes) and CD68 or neutrophil elastase-positive cells by streptavidin-conjugated Alexa 633 (Molecular Probes). Single stainings of each marker were performed to assure specificity of staining patterns in the double stainings. For evaluation, the Leica confocal scanner TCS2 AOBS with an inverted Leica DMIRE2 microscope was used.

Statistics

Data were analyzed statistically with an unpaired, two-tailed Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
M and M-like proteins trigger the release of IL-6 in human blood

IL-6 is an inflammatory mediator with an important role in the pathogenesis of infectious diseases. Importantly, the release pattern of IL-6 correlates with that of other inflammatory markers such as C3a, lactate, and C-reactive protein (12, 35). Therefore, we used IL-6 as a marker of inflammation when testing the effects of various M or M-like proteins in our ex vivo model. Fig. 1A shows that incubations of M1 protein or protein H (both proteins are from an S. pyogenes isolate of the M1 serotype) with human blood result in an IL-6 response comparable with that evoked by LPS. Similar findings were obtained when the effect of other M or M-like proteins with a predicted {alpha}-helical coiled coil structure was investigated. Thus, M proteins of serotypes M3, M5, and M49 and protein FOG from group G streptococci also induced secretion of IL-6. However, surface-associated proteins lacking this overall structure, i.e., protein A (Staphylococcus aureus), protein G (group C and G streptococci), and protein PAB (Finegoldia magna), or proteins that are secreted by S. pyogenes (protein SIC and SpeB) induced no or low IL-6 secretion. As the M1 protein is well characterized and S. pyogenes strains of the M1 serotype are commonly isolated from patients with severe invasive disease (14), this protein was used throughout this study. Thus, all additional experiments were conducted with M1 protein that was purified from overnight cultures of an isogenic M1 mutant strain, termed MC25, expressing a truncated M1 protein lacking the C-terminal cell wall-anchoring motif (20). The effect of MC25-derived M1 protein on IL-6 release was dose dependent (Fig. 1B). Heat inactivation of M1 protein completely abolished its effect on IL-6 release, and trypsin digestion of the protein reduced its stimulatory potential >50% (data not shown). To further ensure that the M1 protein preparation was free of contaminants, we applied overnight cultures from another M1 mutant strain, that lacks M1 protein and protein H (36), to the same purification protocol. This sample, in the following referred to as control preparation, was free of M1 protein as confirmed by ELISA, and it did not trigger IL-6 release when added to whole blood (data not shown). These experiments rule out the possibility that contamination in the M1 protein preparation was responsible for the induction of IL-6 release.


Figure 1
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  		FIGURE 1. IL-6 release in human blood induced by bacterial proteins. A, Human blood was diluted 1/10 in PBS and incubated with M1 protein, protein H (PH), protein SIC, SpeB, the M proteins (M3, M5, and M49), protein FOG, protein A (PA), protein G (PG), or protein PAB at a final concentration of 1 µg/ml. LPS (100 ng/ml) served as a positive control. After incubation for 24 h at 37°C, cells were pelleted, and supernatants were analyzed for IL-6 content by ELISA. B, Human blood treated with different concentrations of M1 protein (———) and LPS (– – – –). Whole blood samples from different human donors were incubated with FITC-labeled M1 protein (C) or HBP (D), as described in Materials and Methods, and analyzed by flow cytometry. Cells were gated based on FSC/SSC characteristics; values represent means ± SD of three experiments.

 
M1 protein triggers monocytes to release cytokines in synergy with HBP

To identify which cell type(s) in human blood interacts with M1 protein, the protein was FITC labeled and incubated with blood for 1 h, followed by a lysing step to remove the erythrocyte fraction. The white blood cell population was then subjected to flow cytometry and analyzed for M1 protein binding. The results show that most of the M1 proteins were bound to monocytes and some to PMNs, whereas only a minor fraction interacted with the lymphocyte population (Fig. 1C). Previously, we have reported that complexes formed between M1 protein and fibrinogen trigger PMNs to release HBP (15). Because it has been shown that internalization of HBP by monocytes (37) potentiates the LPS-induced release of IL-6 and TNF-{alpha} (26), we wished to test whether HBP also binds to monocytes. When FITC-labeled HBP was added to human blood, the protein was found to bind to monocytes, but we also found HBP attached to PMNs and to a lesser extent to the B and T cell populations (Fig. 1D). To investigate whether HBP can modulate the M1 protein-induced cytokine response, purified monocytes were incubated with M1 protein in the absence or presence of HBP. In the absence of HBP, binding of M1 protein to monocytes was followed by the release of IL-6 (1563–3029 pg/ml) into the culture medium, and similar results were recorded when the concentrations of IL-1beta (784–1211 pg/ml) and TNF-{alpha} (261–1354 pg/ml) were measured (Fig. 2A). However, in the presence of HBP, the levels of all secreted cytokines were increased, whereas treatment with HBP alone had no effect (Fig. 2A). These experiments demonstrate that M1 protein interacts with monocytes and that the cytokine release is enhanced in the presence of HBP.


Figure 2
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  		FIGURE 2. IL-6 release induced by M1 protein is enhanced by HBP. A, Purified PBMCs from different donors were stimulated with M1 protein in the presence or absence of HBP. After a 24-h incubation, cells were pelleted, and the release of IL-6, IL-1beta, and TNF-{alpha} was determined. B, Whole blood, diluted 1/10 in PBS, was stimulated with M1 protein in the absence or presence of rabbit IgG against HBP or unrelated rabbit IgG. Cells were pelleted after 24 h, and the levels of IL-6 in the supernatants were determined. Values express cytokine release relative to the response evoked by M1 protein alone, and the bars represent means ± SD of three (A) or four (B) separate experiments. Statistical analysis was performed using Student’s t test (unpaired, two tailed). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
In another set of experiments, we investigated the influence of HBP on M1 protein-mediated cytokine release in whole blood. After an overnight incubation with M1 protein, ~2 µg HBP were released per ml of blood (data not shown). To test whether the released HBP enhances M1 protein-induced IL-6 response, we used neutralizing Abs against HBP. Fig. 2B shows that coincubation of M1 protein with these Abs led to a significant reduction in IL-6 secretion, whereas a control Ab did not influence the M1 protein-induced release of IL-6.

M1 protein interacts with TLR2 on monocytes

The finding that the effect of HBP on the M1 protein-induced pattern of cytokine secretion resembles that of LPS raised the question of whether TLRs are involved in the interaction between M proteins and monocytes. To test this hypothesis, we used CHO cells that were transfected with human CD14/TLR2 or CD14/TLR4. Previous studies have shown that stimulation of these cells by ligands specific for their respective TLR, causes an up-regulation of cotransfected human CD25 (the T cell surface marker IL-2R{alpha}) which is under the control of the NF-{kappa}B-dependent human E-selectin promoter (28). To investigate the effect of M1 protein in this system, CD14/TLR2- and CD14/TLR4-transfected cells were incubated with serial dilutions of the streptococcal protein. Fig. 3A shows that M1 protein triggers an increase in the number of CD25+ CD14/TLR2-transfected cells in a dose-dependent manner, but not of those transfected with CD14/TLR4 (Fig. 3B). Formaldehyde-killed Staphylococcus aureus bacteria and LPS were used to analyze the efficacy of CD14/TLR2 and CD14/TLR4 transfection, respectively, and protein PAB served as a negative control (Fig. 3, A and B). When using TLR2-transfected HEK 293 cells, a dose-dependent activation of the receptor by M1 protein was also observed (Fig. 3C). The control preparation did not activate these cells (Fig. 3C), and cells that were transfected with plasmids containing the genes for NF-{kappa}B reporter luciferase and Rous sarcoma virus-beta-galactosidase but not for TLR2 did not respond to M1 protein treatment (data not shown). Together with the findings in CHO cells, these data show that activation by M1 protein involves TLR2.


Figure 3
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  		FIGURE 3. M1 protein activates TLR-transfected cells via TLR2. CHO cells transfected with CD14 and TLR2 (A) or CD14 and TLR4 (B) were stimulated overnight with different concentrations of M1 protein, PAB (1 µg/ml), LPS (0.5 µg/ml), or formaldehyde-inactivated Staphylococcus aureus. Activated cells were defined by the up-regulation of CD25 as detected by flow cytometry after staining with RPE-conjugated anti-CD25 mAb. Values are the means ± SD of two separate experiments, each done in duplicate. C, HEK 293 cells were cultured overnight at a density of 105 cells/well in 12-well tissue culture plates, followed by transfection with plasmids encoding for beta-galactosidase, endothelial leukocyte adhesion molecule NF-{kappa}B reporter, and human TLR2. Twenty-four hours after transfection, cells were stimulated with M1 protein or the control preparation for 20 h, followed by measurement of cellular activation using chemiluminescence. Results are expressed as the ratio of luciferase to beta-galactosidase and are the means ± SD of experiments performed in triplicates. Values are representative of at least three separate experiments.

 
To visualize the interaction of M1 protein with TLR2 by electron microscopy, the streptococcal protein was incubated with human monocytes for 30 min. Cells were fixed, thin sectioned, and stained with gold-labeled Abs against M1 protein (15 nm in diameter) and TLR2 (4 nm in diameter). Subsequent immunoelectron microscopy analysis revealed that M1 protein was almost exclusively found attached to the cell membrane and frequently colocalized with TLR2 (Fig. 4). Taken together, these results provide evidence that M1 protein triggers cytokine release in monocytes by interacting with TLR2.


Figure 4
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  		FIGURE 4. Colocalization of M1 protein and TLR2 on human monocytes. Ultrathin sections were incubated with 15-nm gold-labeled anti-M1 protein Abs and 4-nm gold-labeled TLR2 Abs and processed as described in Materials and Methods. Colocalization of M1 protein (arrows) and TLR2 (arrowheads) is visible at the plasma membrane. Inset, magnified area containing a complex. Bar, 0.2 µm.

 
HBP activates monocytes via CD11/CD18

The binding of HBP to PMNs is mediated by CD11/CD18 (also known as CR3, Mac-1, or {alpha}Mbeta2; Ref. 38), an integrin that is also expressed on monocytes and activated lymphocytes. Binding to CD11/CD18 in these cells leads to the induction of various intracellular kinases such as the src family tyrosine kinases and tyrosine kinases of the focal adhesion kinase family, which eventually results in the mobilization of intracellularly stored calcium (39). To test whether HBP is able to activate monocytes via binding to CD11/CD18, the calcium response in MM6 cells, a monocytic cell line, was measured. Fig. 5 shows that incubation of MM6 cells with HBP caused an increase of intracellular levels of free Ca2+. This increase was brought to background levels when cells were coincubated with a blocking mAb against beta2 integrins (IB4) (38), whereas an isotype control Ab had no effect (Fig. 5A). Similar results were recorded when HBP was added in the presence of a fibrinogen-derived peptide (Gly-Pro-Arg-Pro; data not shown) that inhibits adherence of PMNs to fibrinogen-coated surfaces (40). In contrast, a control peptide from the same region (Gly-His-Arg-Pro) that does not interact with beta2 integrins had no effect. Coincubation with M1 protein did not influence the HBP-induced mobilization of calcium (data not shown), nor did treatment with M1 protein alone evoke a calcium signal (Fig. 5B). To address the influence of HBP on the M1 protein signaling pathway, brefeldin A was used. Brefeldin A is a fungal-derived macrocyclic lactone inhibitor of protein trafficking in the endomembrane system of mammalian cells that inhibits secretion and vacuolar protein transport (41). It has been shown that LPS-activated monocytes treated with brefeldin A are still able to synthesize IL-6, IL-1beta, and TNF-{alpha}, but due to the impaired exocytosis machinery, these cytokines are not secreted; thus, they accumulate intracellularly (42). Accordingly, we observed an intracellular increase of these cytokines in brefeldin A-treated monocytes stimulated with M1 protein (Fig. 6). However, the effect of M1 protein was not potentiated by HBP. Taken together, the results show that M1 protein and HBP use different signaling pathways. The brefeldin A experiments suggest that HBP does not induce an additional increase in protein synthesis. However, the exact mechanism behind the effect of HBP is still not completely understood.


Figure 5
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  		FIGURE 5. HBP induces intracellular Ca2+ mobilization in MM6 cells. A, Dynamic change in fluorescence intensity of MM6 cells loaded with fluo-4/AM after stimulation with 500 ng/ml HBP ({cjs2108}), buffer ({diamond}), 500 ng/ml HBP in the presence of 10 µg/ml IB4 ({blacktriangleup}), or 500 ng/ml HBP in the presence of 10 µg/ml isotype control IgG ({triangleup}). Cells were pretreated with the Abs 30 min before the addition of HBP. B, Dynamic change in fluorescence intensity of MM6 cells loaded with fluo4/AM after stimulation with 500 ng/ml HBP ({cjs2108}), buffer ({diamond}), or 1 µg/ml M1 protein (•). Measurements were made by flow cytometry in MM6 cell suspensions before and immediately after stimulation and then at 30-s intervals. Values are expressed in percent of mean fluorescence intensity before treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 from sham treatment value. n = 5. Values are means ± SD.

 

Figure 6
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  		FIGURE 6. HBP does not affect cytokine production of brefeldin A-treated monocytes. Human PBMCs treated with brefeldin A were stimulated with M1 protein in the absence or presence of HBP. Cells were subsequently stained with RPE-conjugated anti-CD14 and/or FITC-conjugated anti-IL-6, anti-IL-1beta, or anti-TNF-{alpha} mAbs, as described in Materials and Methods, and analyzed by flow cytometry. As controls, cells were incubated with brefeldin A alone or together with HBP. Shown are the monocyte population gated on FSC/SSC characteristics and CD14 expression. Bars represent means ± SD of three experiments.

 
Analysis of patient tissue biopsies reveals that HBP is released from PMNs and taken up by monocytes/macrophages

Previous in vitro studies have shown that M1 protein/fibrinogen complexes activate PMNs to release HBP and that M1 protein is solubilized from the bacterial surface at the site of infection in vivo, where it penetrates the tissue (15). Here, analyses of tissue biopsies from patients with necrotizing fasciitis or severe cellulitis caused by S. pyogenes bacteria (M1 serotype) revealed that the recruitment of PMNs and monocytes/macrophages to the infectious focus is accompanied by the release of HBP (Fig. 7, A–D). Quantitation of bacterial load, cell infiltration, and expression of HBP in the tissue was achieved by ACIA (Fig. 7E), and all markers were found to be considerably higher than in a tissue biopsy from the noninfected leg of a patient with streptococcal erysipelas (ACIA values: S. pyogenes, 0.12; macrophages, 1.14; neutrophils, 0.18; HBP, 2.1, respectively). In addition, confocal microscopy showed that PMNs had emptied their intracellular HBP storage, and large amounts of HBP are seen outside the PMNs diffusing into the tissue (Fig. 7, F and G). Some of the released HBP is also taken up by monocytes/macrophages at the site of infection (Fig. 7, H and I). These findings further underline the significance of interactions among M1 protein, PMNs, HBP, and monocytes/macrophages in the infectious-inflammatory processes during severe S. pyogenes infection.


Figure 7
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  		FIGURE 7. Analysis of tissue biopsies from patients with necrotizing fasciitis and severe cellulites. A—D, Presence of bacteria and dense infiltration of PMNs and monocytes/macrophages at the local site of infection. Tissue biopsies (n = 8) collected at the site of infection from three patients with necrotizing fasciitis or severe cellulitis caused by M1T1 group A streptococci were stained for the presence of bacteria (A), monocytes/macrophages (B), PMNs (C), and HBP (D). A—D, Stainings of a representative biopsy. All stained biopsies were evaluated by ACIA, and the ACIA values for each marker are shown in E. The tissue biopsies were also analyzed by confocal microscopy for evaluation of the localization of HBP (Alexa 488, green) in relation to PMNs (Alexa 633, magenta), and monocytes/macrophages (Alexa 633, magenta; F–I). Shown is a maximum projection of a sequential scan. Cellular infiltrates are indicated in blue by the nuclear stain DAPI. A–D and F—I, stainings of a representative biopsy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The intensity of the host response to a bacterial infection is an important parameter that determines the outcome of the disease (43). Thus, in severe infectious diseases, an overreaction of the host may lead to systemic activation of inflammatory pathways including cytokine induction and activation of the complement and coagulation systems. S. pyogenes is an important Gram-positive pathogen that causes powerful inflammatory reactions, which in severe cases may lead to life-threatening complications such as disturbed microcirculation, hypotension, shock, and organ failure (44, 45). Recently we reported that M protein released from the bacterial surface, forms complexes with fibrinogen that trigger the secretion of HBP, a potent inducer of vascular leakage (30, 46), from human PMNs (15). In this investigation, we find that in addition to interactions with PMNs, M protein is also a potent activator of monocytes. However, the mechanism of activation is different and involves TLR2. The interaction between M1 protein and TLR2 on monocytes results in cytokine production, especially IL-6. In this context, it is noteworthy that IL-6 levels in the blood of patients with severe S. pyogenes infections can reach extremely high concentrations (>10 µg/ml; Ref. 47), and that, as shown here, the cytokine response is enhanced in the presence of HBP. Because PMNs are usually the first cells to be recruited to the site of infection, followed by monocytes, M proteins could have a dual function in the induction of inflammatory reactions at the site of infection, in the first step by inducing the release of HBP from invading PMNs which, in a second step, enhances M protein-triggered secretion of proinflammatory cytokines from monocytes (Fig. 8). Support for this hypothesis was obtained through analyses of biopsies from patients with severe streptococcal soft tissue infection in which interaction among group A streptococci, neutrophils, macrophages, and HBP could be demonstrated. Also in systemic infections, S. pyogenes bacteria and soluble M protein in the blood stream could induce a deleterious release of cytokines by a similar mode of action. The findings of the present work are in line with other studies showing that LPS-induced induction of proinflammatory cytokines is enhanced in the presence of HBP (26).


Figure 8
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  		FIGURE 8. Schematic model for M protein-induced inflammation. During S. pyogenes infection, M protein is released from the bacterial surface spontaneously, or via host- and bacteria-derived proteinases. Released M1 protein forms complexes with fibrinogen that trigger PMNs to release HBP. M protein can also directly activate monocytes by interacting with TLR2, resulting in the secretion of proinflammatory cytokines. Binding of HBP to beta2 integrins of monocytes potentiates M protein-induced inflammation and enhances cytokine secretion.

 
Given that TLR2 has a broad specificity for the recognition of pathogen-associated molecular patterns from different microorganisms including also mycobacteria, protozoa, and fungi (48), the receptor is an interesting drug target. This notion is supported by a recent report showing that an antagonistic Ab against TLR2 was protective in a mouse model of Bacillus subtilis sepsis (49). Thus, the Ab down-regulated the levels of IL-6 and TNF-{alpha} in the serum of mice treated with the TLR2 agonist P3CSK4 (49). In the present study, we find that a similar down-regulation of proinflammatory cytokines can be obtained in M1 protein-treated human blood by blocking HBP with an Ab. These results imply that the development of antagonists targeting TLR2 or HBP could lead to novel therapeutic strategies in severe streptococcal infections.

In summary, the present study has identified a novel mechanism by which S. pyogenes induces a massive inflammatory response, involving streptococcal M protein, PMN-derived HBP, and TLR2 on monocytes. The results help to explain the extremely fast progression of this devastating disease.


   Acknowledgments
 
We thank Monica Heidenholm and Maria Baumgarten (Department of Clinical Sciences, Lund University) and Marta Brant (Department of Laboratory Medicine, Mälmo University Hospital) for excellent technical assistance; Hans Flodgaard (Leukotech) for providing HBP and Abs against HBP; and Rita Wallén and Eric Hallberg (Cell and Organism Biology, Lund University) and Lina Gefors (Jubileum Institute, Lund University) for support with electron microscopy.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported in part by the foundations of Alfred Österlund, Anna and Edwin Berger, Clas Groschinsky, Crafoord, Greta and Johan Kock, and Åke Wiberg; the Swedish Foundation for Strategic Research; the Swedish Heart-Lung Foundation; the AFA Health Fund; King Gustaf V’s 80-Years Fund; the Royal Physiographical Society in Lund; the Odd Fellow Sweden; the Blood and Defence Network and the Vascular Wall Programme at Lund University; the Medical Faculty, Lund University; the Swedish Research Council (Projects 4342, 7480, and 13413); the Deutsche Forschungsgemeinschaft (Innate Immunity, Project Schr. 726, 1-2 and SFB 633-03, Project A7); and Hansa Medical AB. Back

2 Current address: AstraZeneca R&D Lund, SE-22187 Lund, Sweden. Back

3 Address correspondence and reprint requests to Dr. Heiko Herwald, Department of Clinical Sciences, Section for Clinical and Experimental Infection Medicine, Biomedicinskt Centrum, B14, Lund University, Tornavägen 10, SE-221 84 Lund, Sweden. E-mail address: Heiko.Herwald{at}med.lu.se Back

4 Abbreviations used in this paper: PMNs , polymorphonuclear neutrophils; PRR, pattern recognition receptors; HBP, heparin-binding protein; MM6, Mono Mac 6; HEK, human embryonic kidney; FSC, forward scatter; SSC, side scatter; CHO, Chinese hamster ovarian; ACIA, acquired computerized image analysis. Back

Received for publication December 5, 2005. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Athman, R., D. Philpott. 2004. Innate immunity via Toll-like receptors and Nod proteins. Curr. Opin. Microbiol. 7: 25-32. [Medline]
  2. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  3. Hoebe, K., E. Janssen, B. Beutler. 2004. The interface between innate and adaptive immunity. Nat. Immunol. 5: 971-974. [Medline]
  4. Gordon, S.. 2002. Pattern recognition receptors: doubling up for the innate immune response. Cell. 111: 927-930. [Medline]
  5. Philpott, D. J., S. E. Girardin. 2004. The role of Toll-like receptors and Nod proteins in bacterial infection. Mol. Immunol. 41: 1099-1108. [Medline]
  6. Akira, S., S. Sato. 2003. Toll-like receptors and their signaling mechanisms. Scand. J. Infect. Dis. 35: 555-562. [Medline]
  7. Beutler, B.. 2004. Innate immunity: an overview. Mol. Immunol. 40: 845-859. [Medline]
  8. Takeda, K., S. Akira. 2003. Toll receptors and pathogen resistance. Cell. Microbiol. 5: 143-153. [Medline]
  9. Yamamoto, M., K. Takeda, S. Akira. 2004. TIR domain-containing adaptors define the specificity of TLR signaling. Mol. Immunol. 40: 861-868. [Medline]
  10. Iwasaki, A., R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987-995. [Medline]
  11. Tsan, M. F., B. Gao. 2004. Endogenous ligands of Toll-like receptors. J. Leukocyte Biol. 76: 514-519. [Abstract/Free Full Text]
  12. Cavaillon, J. M., M. Adib-Conquy, C. Fitting, C. Adrie, D. Payen. 2003. Cytokine cascade in sepsis. Scand. J. Infect. Dis. 35: 535-544. [Medline]
  13. Stevens, D. L.. 2003. Group A streptococcal sepsis. Curr. Infect. Dis. Rep. 5: 379-386. [Medline]
  14. Cunningham, M. W.. 2000. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13: 470-511. [Abstract/Free Full Text]
  15. Herwald, H., H. Cramer, M. Mörgelin, W. Russell, U. Sollenberg, A. Norrby-Teglund, H. Flodgaard, L. Lindbom, L. Björck. 2004. M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage. Cell 116: 367-379. [Medline]
  16. Lancefield, R. C.. 1969. Current problems in studies of streptococci. J. Gen. Microbiol. 55: 161-163. [Free Full Text]
  17. Fischetti, V. A.. 1989. Streptococcal M protein: molecular design and biological behaviour. Clin. Microbiol. Rev. 2: 285-314. [Abstract/Free Full Text]
  18. Åkesson, P., K. H. Schmidt, J. Cooney, L. Björck. 1994. M1 protein and protein H: IgGFc- and albumin-binding streptococcal surface proteins encoded by adjacent genes. Biochem. J. 300: 877-886. [Medline]
  19. Berge, A., L. Björck. 1995. Streptococcal cysteine proteinase releases biologically active fragments of streptococcal surface proteins. J. Biol. Chem. 270: 9862-9867. [Abstract/Free Full Text]
  20. Collin, M., A. Olsén. 2000. Generation of a mature streptococcal cysteine proteinase is dependent on cell wall-anchored M1 protein. Mol. Microbiol. 36: 1306-1318. [Medline]
  21. Weineisen, M., U. Sjöbring, M. Fallman, T. Andersson. 2004. Streptococcal M5 protein prevents neutrophil phagocytosis by interfering with CD11b/CD18 receptor-mediated association and signaling. J. Immunol. 172: 3798-3807. [Abstract/Free Full Text]
  22. von Pawel-Rammingen, U., B. P. Johansson, L. Björck. 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J. 21: 1607-1615. [Medline]
  23. Johansson, H. M., M. Mörgelin, I. M. Frick. 2004. Protein FOG: a streptococcal inhibitor of neutrophil function. Microbiology 150: 4211-4221. [Abstract/Free Full Text]
  24. de Château, M., L. Björck. 1994. Protein PAB, a mosaic albumin-binding bacterial protein representing the first contemporary example of module shuffling. J. Biol. Chem. 269: 12147-12151. [Abstract/Free Full Text]
  25. Frick, I. M., P. Å kesson, M. Rasmussen, A. Schmidtchen, L. Björck. 2003. SIC, a secreted protein of Streptococcus pyogenes that inactivates antibacterial peptides. J. Biol. Chem. 278: 16561-16566. [Abstract/Free Full Text]
  26. Rasmussen, P. B., S. Bjørn, S. Hastrup, P. F. Nielsen, K. Norris, L. Thim, F. C. Wiberg, H. Flodgaard. 1996. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS induced cytokine release from monocytes. FEBS Lett. 390: 109-112. [Medline]
  27. Ziegler-Heitbrock, H. W., E. Thiel, A. Futterer, V. Herzog, A. Wirtz, G. Riethmüller. 1988. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int. J. Cancer 41: 456-461. [Medline]
  28. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163: 1-5. [Abstract/Free Full Text]
  29. Lazou Ahrén, I., A. Bjartell, A. Egesten, K. Riesbeck. 2001. Lipopolysaccharide-binding protein increases toll-like receptor 4-dependent activation by nontypeable Haemophilus influenzae. J. Infect. Dis. 184: 926-930. [Medline]
  30. Gautam, N., A. M. Olofsson, H. Herwald, L. F. Iversen, E. Lundgren-Åkerlund, P. Hedqvist, K. E. Arfors, H. Flodgaard, L. Lindbom. 2001. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat. Med. 7: 1123-1127. [Medline]
  31. Baschong, W., N. G. Wrigley. 1990. Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution labels for electron microscopy: a technical overview. J. Electron Microsc. Tech. 14: 313-323. [Medline]
  32. Roth, J.. 1986. Post-embedding cytochemistry with gold-labelled reagents: a review. J. Microsc. 143: 125-137. [Medline]
  33. Norrby-Teglund, A., P. Thulin, B. S. Gan, M. Kotb, A. McGeer, J. Andersson, D. E. Low. 2001. Evidence for superantigen involvement in severe group a streptococcal tissue infections. J. Infect. Dis. 184: 853-860. [Medline]
  34. Lindmark, A., D. Garwicz, P. B. Rasmussen, H. Flodgaard, U. Gullberg. 1999. Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/azurocidin. J. Leukocyte Biol. 66: 634-643. [Abstract]
  35. Hack, C. E., E. R. De Groot, R. J. Felt-Bersma, J. H. Nuijens, R. J. Strack Van Schijndel, A. J. Eerenberg-Belmer, L. G. Thijs, L. A. Aarden. 1989. Increased plasma levels of interleukin-6 in sepsis. Blood 74: 1704-1710. [Abstract/Free Full Text]
  36. Kihlberg, B. M., J. Cooney, M. G. Caparon, A. Olsén, L. Björck. 1995. Biological properties of a Streptococcus pyogenes mutant generated by Tn916 insertion in mga. Microb. Pathog. 19: 299-315. [Medline]
  37. Heinzelmann, M., A. Platz, H. Flodgaard, H. C. Polk, Jr, F. N. Miller. 1999. Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-{alpha} production in human monocytes. J. Immunol. 162: 4240-4245. [Abstract/Free Full Text]
  38. Cai, T. Q., S. D. Wright. 1996. Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, {alpha}Mbeta2) and modulates polymorphonuclear leukocyte adhesion. J. Exp. Med. 184: 1213-1223. [Abstract/Free Full Text]
  39. Dib, K., T. Andersson. 2000. beta2 integrin signaling in leukocytes. Front. Biosci. 5: D438-D451. [Medline]
  40. Loike, J. D., B. Sodeik, L. Cao, S. Leucona, J. I. Weitz, P. A. Detmers, S. D. Wright, S. C. Silverstein. 1991. CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A{alpha} chain of fibrinogen. Proc. Natl. Acad. Sci. USA 88: 1044-1048. [Abstract/Free Full Text]
  41. Nebenfuhr, A., C. Ritzenthaler, D. G. Robinson. 2002. Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol. 130: 1102-1108. [Free Full Text]
  42. Schuerwegh, A. J., W. J. Stevens, C. H. Bridts, L. S. De Clerck. 2001. Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1beta, interleukin-6, and tumor necrosis factor-{alpha} in monocytes. Cytometry 46: 172-176. [Medline]
  43. Cohen, J.. 2002. The immunopathogenesis of sepsis. Nature 420: 885-891. [Medline]
  44. Nowak, R.. 1994. Flesh-eating bacteria: not new, but still worrisome. Science 264: 1665[Free Full Text]
  45. Stevens, D. L.. 2000. Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annu. Rev. Med. 51: 271-288. [Medline]
  46. Edens, H. A., C. A. Parkos. 2003. Neutrophil transendothelial migration and alteration in vascular permeability: focus on neutrophil-derived azurocidin. Curr. Opin. Hematol. 10: 25-30. [Medline]
  47. Holub, M., M. Helcl, O. Beran, J. Hnyková, J. Príhodová. 2004. Kinetics of immune parameters in a patient with sepsis caused by Streptococcus pyogenes treated with activated protein C. Scand. J. Infect. Dis. 36: 485-488. [Medline]
  48. Kirschning, C. J., R. R. Schumann. 2002. TLR2: cellular sensor for microbial and endogenous molecular patterns. Curr. Top. Microbiol. Immunol. 270: 121-144. [Medline]
  49. Meng, G., M. Rutz, M. Schiemann, J. Metzger, A. Grabiec, R. Schwandner, P. B. Luppa, F. Ebel, D. H. Busch, S. Bauer, H. Wagner, C. J. Kirschning. 2004. Antagonistic antibody prevents Toll-like receptor 2-driven lethal shock-like syndromes. J. Clin. Invest. 113: 1473-1481. [Medline]

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