Holger Heine

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Bacterial Endotoxin: Chemical Constitution, Biological Recognition, Host Response, and Immunological Detoxification

The discovery of endotoxin dates from the late nineteenth century when Richard Pfeiffer, then working in Berlin, characterized endotoxins as heat-stable and cell-associated molecules (Westphal et al. 1977), thus distinguishing them from the heat-labile and proteinous exotoxins which are actively secreted by bacteria (Bhakdi et al. 1994). They were first found to be produced by Vibrio cholerae bacteria and later by Salmonella and Serratia. Endotoxins, due to their various potent biological activities soon attracted worldwide scientific interest. Initial chemical analyses of purified endotoxin indicated that it consists essentially of polysaccharide and lipid, and it was therefore termed lipopolysaccharide (LPS). Today the terms endotoxin (Wolff 1904) and lipopolysaccharide (Shear and Turner 1943) are used synonymously for the same molecule.

TLR2 -promiscuous or specific? A critical re-evaluation of a receptor expressing apparent broad specificity

Of all pattern recognition receptors (PRR) in innate immunity, Toll-like receptor 2 (TLR2) recognizes the structurally broadest range of different bacterial compounds known as pathogen-associated molecular patterns (PAMPs). TLR2 agonists identified so far are lipopolysaccharides (LPSs) from different bacterial strains, lipoproteins, (synthetic) lipopeptides, lipoarabinomannans, lipomannans, glycosylphosphatidylinositol, lipoteichoic acids (LTA), various proteins including lipoproteins and glycoproteins, zymosan, and peptidoglycan (PG). Because these molecules are structurally diverse, it seems unlikely that TLR2 has the capability to react with all agonists to the same degree. The aim of this review is to identify and describe well-defined structure-function relationships for TLR2. Because of its biomedical importance and because its genetics and biochemistry are presently most completely known among all Gram-positive bacteria, we have chosen Staphylococcus aureus as a focus. Our data together with those reported by other groups reveal that only lipoproteins/lipopeptides are sensed at physiologically concentrations by TLR2 at picomolar levels. This finding implies that the activity of all other putative bacterial compounds so far reported as TLR2 agonists was most likely due to contaminating highly active natural lipoproteins and/or lipopeptides.

Molecular mechanisms of endotoxin activity

Endotoxin (lipopolysaccharide, LPS), a constitutent of the outer membrane of the cell wall of gramnegative bacteria, exerts a wide variety of biological effects in humans. This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intra-cellular signal transduction pathways.

TLR1- and TLR6-independent recognition of bacterial lipopeptides.

Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides are assumed to be recognized by TLR2/TLR1-, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. Following our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides (e.g. Pam2C-SK4, Pam3C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct2C-SSNASK4), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent manner.

Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling.

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen‐associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up‐regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1‐ or TLR6‐dependent manner. There are also di‐ and triacylated LP, which stimulate TLR1‐deficient cells and TLR6‐deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)2C‐(VPGVG)4VPGKG, fibroblast‐stimulating LP‐1, and Pam2C‐SK4. Dominant‐negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram‐positive and Gram‐negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

Toll-like receptor 6-independent signaling by diacylated lipopeptides.

Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll‐like receptor 2 (TLR2)–TLR1 or TLR2–TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteine (Pam3)‐CSK4, signal through TLR2–TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage‐activating lipopeptide from Mycoplasma fermentans (MALP2) or S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteine (Pam2)‐CGNNDESNISFKEK, induce signaling through TLR2–TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK‐SK4 (MALP2‐SK4) induced B lymphocyte proliferation and TNF‐α secretion in macrophages in a TLR6‐independent manner as determined with cells from TLR6‐deficient mice. Our results indicate that both the lipid and the N‐terminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand–receptor interaction on host cells.

TLR4-mediated inflammatory activation of human coronary artery endothelial cells by LPS

OBJECTIVE Blood levels of cytokines are commonly elevated in severe congestive heart failure (CHF) and in coronary artery disease (CAD). While the adverse effects of cytokines on contractile function and myocardial cell integrity are well studied, little is known on whether cardiac cells are only targets or active players in these inflammatory reactions. METHODS AND RESULTS We tested if human coronary artery endothelial cells (HCAEC) may become a source of cytokine and adhesion molecule expression when stimulated with bacterial lipopolysaccharide (LPS). Analysis of HCAEC supernatants by ELISA identified enhanced secretion of IL-6, IL-8, and MCP-1 while endothelin-1 was not increased. IL-1beta, IL-10, or TNF-alpha were not detectable by ELISA while RT-PCR revealed enhanced mRNA expression of IL-1beta and TNF-alpha but not IL-10. FACS analysis showed an LPS-induced upregulation of ICAM-1, VCAM, and ELAM-1. LFA-1 could not be detected. We further characterized receptors involved in LPS-induced signaling. Our results indicate that activation of HCAEC by LPS requires Toll-like receptor (TLR) 4. Pretreating the cells with the 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitor Cerivastatin reduced IL-6 release. CONCLUSIONS Taken together, our results indicate that activated HCAEC may act as inflammatory cells and thus directly contribute to the progression of CHF and CAD.

Zinc Signals Are Essential for Lipopolysaccharide-Induced Signal Transduction in Monocytes

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn2+), but this still awaits experimental verification. Zn2+ is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-α. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn2+ signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam3CSK4, TNF-α, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N′,N′-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-κB, and abrogates the release of proinflammatory cytokines, including TNF-α. This function of Zn2+ is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca2+.

Toll-Like Receptors and Their Function in Innate and Adaptive Immunity

Over the past 3 years our knowledge about how we sense the microbial world has been fundamentally changed. It has been known for decades that microbial products, such as lipopolysaccharide, lipoproteins, or peptidoglycan, have a profound activity on human cells. Whereas the structure of many different pathogenic microbial compounds has been extensively studied and characterized, the molecular basis of their recognition by the cells of the innate immune system remained elusive for a long time. It was Charles Janeway [Cold Spring Harb Symp Quant Biol 1989;54/1:1–13] who developed the idea of microbial structures forming pathogen-associated molecular patterns that would be recognized by pattern recognition receptors. The discovery of the family of Toll receptors in species as diverse as Drosophila and humans, and the recognition of their role in distinguishing molecular patterns that are common to microorganisms have led to a renewed appreciation of the innate immune system. Moreover, it is now clear that the activation of the innate immune system through mammalian Toll-like receptors has also an instructive role for the responses of the adaptive immune response and, thus, may influence allergic diseases such as asthma.