Artur J. Ulmer

Lipopolysaccharide and ceramide docking to CD14 provokes ligand‐specific receptor clustering in rafts

The glycosylphosphatidylinositol‐anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co‐receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcγ‐receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co‐clustering with Toll‐like receptor 4, Fcγ‐RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand‐specific cellular responses initiated by CD14.

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.

Human lung cancer cells express functionally active Toll-like receptor 9

BackgroundCpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.MethodsThe expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.ResultsWe found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.ConclusionsHere we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

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.

The chemical structure of bacterial endotoxin in relation to bioactivity

Lipopolysaccharides (LPS) constitute the O-antigens and endotoxins of Gram-negative bacteria. Whereas both the polysaccharide and lipid portion of LPS contribute to the pathogenic potential of this class of bacteria, it is the lipid component (lipid A) which determines the endotoxic properties of LPS. The primary structure of lipid A of various bacterial origin has been elucidated and Escherichia coli lipid A has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique structural arrangement and conformation. Such analyses have furthermore provided insight into the determinants required for lipid A binding to and activation of human target cells. Present research efforts aim at the molecular characterization of the specificity, modulation and biomedical consequences of the interaction of lipid A with host cells.

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.

Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG‐labeled derivative of the synthetic lipopeptide N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2R,S)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine (Pam3CSK4) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam3CSK4‐FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam3CSK4‐FLAG binding, CD14 and Pam3CSK4‐FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low‐mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.

Lipopeptide structure determines TLR2 dependent cell activation level

Bacterial lipoproteins/peptides are composed of di‐O‐acylated‐S‐(2,3‐dihydroxypropyl)‐cysteinyl residues N‐terminally coupled to distinct polypeptides, which can be N‐acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester‐bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide‐bound and another ester‐bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno‐stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.