Biomedical subjects

Siegfried Morath

Publications and source records attributed to Siegfried Morath.

25 records · Page 2Linked to original sources

PubMed2003

Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved.

Lipoteichoic acid (LTA) derived from Streptococcus pneumoniae, purified employing a chloroform/methanol protocol, and from Staphylococcus aureus, prepared by the recently described butanol extraction procedure, was investigated regarding its interaction with lipopolysaccharide (LPS)-binding protein (LBP), CD14, Toll-like receptors (TLRs)-2 and -4, and MD-2. LTA from both organisms induced cytokine synthesis in human mononuclear phagocytes. Activation was LBP- and CD14-dependent, and formation of complexes of LTA with LBP and soluble CD14 as well as catalytic transfer of LTA to CD14 by LBP was verified by PhastGel(TM) native gel electrophoresis. Human embryonic kidney (HEK) 293/CD14 cells and Chinese hamster ovary (CHO) cells were responsive to LTA only after transfection with TLR-2. Additional transfection with MD-2 did not affect stimulation of these cells by LTA. Our data suggest that innate immune recognition of LTA via LBP, CD14, and TLR-2 represents an important mechanism in the pathogenesis of systemic complications in the course of infectious diseases brought about by the clinically most important Gram-positive pathogens. However, the involvement of TLR-4 and MD-2 in this process was ruled out.

Acute-Phase Proteins↗

PubMed2003

Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment.

Lipoteichoic acid (LTA) is a major immunostimulatory principle of Gram-positive bacteria. Intranasal application of LTA from S. aureus to mice resulted in greatly increased neutrophil and macrophage counts in the bronchoalveolar lavage as well as increased levels of the chemokine KC. The potential of highly pure, bioactive LTA from S. aureus to induce neutrophil recruitment and activation was investigated further in the human system. Although neutrophils expressed the key known receptors, CD14, TLR2 and TLR6, LTA did not induce or prime neutrophils for oxidative burst, or release of chemokines, bactericidal permeability-increasing protein or myeloperoxidase. However, LTA induced a strong release of the chemoattractants LTB4, IL-8, C5a, MCP-1 and the colony-stimulating factor G-CSF in whole blood comparable to stimulation with the same concentration of LPS (S. abortus equi). Further, the cytokine and chemoattractant pattern induced by LTA correlated well with that induced by live S. aureus of the same strain. LTA does not appear to activate neutrophils directly, but is a strong stimulus for the recruitment of phagocytes to the site of infection.

Animals↗

PubMed2002

Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.

We recently purified lipoteichoic acid (LTA) from Staphylococcus aureus to more than 99% purity by a novel preparation method and deduced its structure with the first nuclear magnetic resonance (NMR) of a complete LTA. In contrast to Gram-negative lipopolysaccharides, this LTA requires the toll-like receptor (TLR)-2 and not TLR-4 for cytokine induction in monocytes and macrophages. To elucidate the structure-function relationships for LTA from S. aureus, the lipid anchor was prepared by either acidic hydrolysis of native LTA or chemical synthesis (gentiobiosyl-sn-dimyristoylglycerol). Next, a complete LTA molecule with six glycerophosphate units carrying four alanine plus one N-acetyl-glucosamine substituent was synthesized, which displayed the same potency to activate monocytes as native LTA. However, 100-1,000 times higher concentrations of the lipid anchor were required for cytokine induction. It is worthy to note that replacing D-alanine with L-alanine blunted the effect indicating stereoselective recognition. The structure identification of this synthesized and biologically active LTA was proven by NMR and matrix-assisted laser desorption-ionization mass spectrometry. We concluded that the lipid anchor, with its fatty acids, represents an integral part of the immunostimulatory activity of LTA, but requires additional structural components on the polyglycerophosphate backbone.

Animals↗

PubMed2002

Cytokine induction by purified lipoteichoic acids from various bacterial species--role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release.

We have recently shown that highly purified lipoteichoic acid (LTA) represents a major immunostimulatory principle of Staphylococcus aureus. In order to test whether this translates to other bacterial species, we extracted and purified LTA from 12 laboratory-grown species. All LTA induced the release of TNF-alpha, IL-1beta, IL-6 and IL-10 in human whole blood. Soluble CD14 (sCD14) inhibited monokine induction by LTA but failed to confer LTA responsiveness for IL-6 and IL-8 release of human umbilical vein endothelial cells (HUVEC). In a competitive LPS-binding protein (LBP) binding assay, the IC(50) of the tested LTA preparations was up to 3,230-fold higher than for LPS. LBP enhanced TNF-alpha release of human peripheral blood mononuclear cells (PBMC) upon LPS but not LTA stimulation. These data demonstrate a differential role for the serum proteins LBP and sCD14 in the recognition of LPS and LTA. Different efficacies of various anti-CD14 antibodies against LPS vs. LTA-induced cytokine release suggest that the recognition sites of CD14 for LPS and LTA are distinct with a partial overlap. While the maximal achievable monokine release in response to LTA was comparable to LPS, all LTA induced significantly less IL-12 and IFN-gamma. IL-12 substitution increased LTA-inducible IFN-gamma release up to 180-fold, suggesting a critical role of poor LTA-inducible IL-12 for IFN-gamma formation. Pretreatment with IFN-gamma rendered galactosamine-sensitized mice sensitive to challenge with LTA. In conclusion, LTA compared to LPS, are weak inducers of IL-12 and subsequent IFN-gamma formation which might explain their lower toxicity in vivo.

Acute-Phase Proteins↗

PubMed2002

Interaction of lipoteichoic acid and CpG-DNA during activation of innate immune cells.

The innate immune system recognizes pathogen-associated molecular patterns (PAMP) to cope with evolving infections. Toll-like receptors (TLRs) play a pivotal role in recognition of PAMPs. In the course of infection not a single but rather a full panel of different microbial components interacts with distinct TLRs simultaneously. Only limited information is available on effects of combinations of TLR agonists. Here, we have analyzed the effects of lipoteichoic acid (LTA), CpG-DNA and combinations thereof on innate immune cells in vitro. Although proinflammatory cytokines like TNF-alpha were induced by these agonists in quite similar amounts, CpG DNA was superior in its potency to induce IL-12p40 reflecting important differences in the biological valence of LTA and CpG-DNA. When given in combination, LTA and CpG-DNA were additive in induction of TNF-alpha, IL-6 and nitric oxide in RAW 264 macrophages, peritoneal macrophages and dendritic cells. Additive effects were also observed in regard to TNF-alpha mRNA. In contrast, LTA suppressed IL12p40 secretion induced by CpG-DNA in RAW cells and peritoneal macrophages but not in dendritic cells. Intracellular signal cascades (NFkappaB and p38 MAP kinase) showed additive effects after simultaneous triggering. mRNA expression ofTLRs showed only minor regulation after CpG or LTA application and thus does not account for the additive/suppressive effects observed. These results indicate that the consequences of interaction of innate immune cells with microbial pattern depend on the responding cell type and might be differential for certain effector mechanisms. Thus, the pathogen-characteristic panel of TLR ligands will induce pathogen-specific innate responses decisive for the inflammatory reactions.

Animals↗

PubMed2002

Structural decomposition and heterogeneity of commercial lipoteichoic Acid preparations.

Fractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% for Streptococcus pyogenes, 16% for Bacillus subtilis, and 75% for Staphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine and N-acetylglucosamine substituents. All preparations contained-to varying degrees-non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA from Bacillus subtilis and S. pyogenes but not LTA from S. aureus induced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.

Bacillus subtilis↗

PubMed2002

Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14.

BACKGROUND: The pro-inflammatory potential of lipoteichoic acid (LTA) from Staphylococcus aureus is controversial. The present study was undertaken to examine the ability of highly purified and characterized S. aureus LTA to stimulate the production of pro-inflammatory cytokines in human leukocytes at both mRNA and protein level, and to study the involvement of Toll-like receptors (TLRs) and CD14 in this response. MATERIAL/METHODS: Purified LTA was administered to whole human blood ex-vivo (or primary adherent monocytes) and the cytokine response assessed in plasma by EIA. Cytokine mRNA was measured by RT-PCR on leukocyte subsets isolated following stimulation. To study the involvement of specific receptors for LTA signaling, CHO cells transfected with CD14 and/or TLR2, TLR4 were used, as well as antibodies directed against these receptors. RESULTS: Addition of highly purified LTA to human whole blood or primary adherent human monocytes elicited a time and concentration dependent release of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6 and IL-8. Messenger RNA encoding TNF-alpha, IL-1 beta and IL-6 seemed to be accumulated in monocytes and T cells, but not in granulocytes and B cells. Expression of TLR2, but not TLR4, in chinese hamster ovary cells conferred responsiveness to LTA. However, antibodies directed towards TLR2 (clone TL2.1) or TLR4 (clone HTA125) failed to inhibit TNF-a release induced by LTA in both the whole blood model and in adherent monocytes. In contrast, blockade of the CD14 receptor with MAb18D11 strongly attenuated the LTA induced release of TNF-alpha in both models. CONCLUSIONS: We propose that (i) LTA from S. aureus triggers the release of cytokines during staphylococcal infections, and (ii) both monocytes and T cells contribute to cytokine production induced by LTA. TLR2 may mediate cellular activation by LTA, but the functional significance of this receptor during staphylococcal infections remains elusive.

Actins↗