Biomedical subjects

Ute Buwitt-Beckmann

Publications and source records attributed to Ute Buwitt-Beckmann.

6 recordsLinked to original sources

PubMed2006

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. Pam(2)C-SK(4), Pam(3)C-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 PamOct(2)C-SSNASK(4)), 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.

Animals↗

PubMed2005

Forming and immunological properties of some lipopolysaccharide-chitosan complexes.

The complex formation of lipopolysaccharide (LPS) with chitosan (Ch) was demonstrated using sedimentation velocity analysis in the analytical ultracentrifuge, centrifugation in glycerol gradient and isopicnic centrifugation in cesium chloride. An addition of Ch to the Escherichia coli and Yersinia pseudotuberculosis LPS solutions was found to result in formation of the stable LPS-Ch complexes. The interaction is a complicated process and depends on time and reaction temperature, as well as on the molecular weight of chitosan. A stable LPS-Ch complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). It should be noted that process of LPS complexation with Ch is accompanied by additional dissociating of LPS. The complex formation was shown to be a result not only of ionic binding, but also of other types of interactions. The interaction of Ch with LPS was shown to modulate significantly the biological activity of LPS. The LPS-Ch complex (1:5 w/w) was shown to possess much lower toxicity in a comparison with the parent LPS at injection to mice in the similar concentration. The LPS-Ch complex was shown to maintain an ability to induce of IL-8 and TNF, but induction of IL-8 and TNF biosynthesis by the LPS-Ch complex was lower than that by the parent LPS. The complex LPS-Ch, similarly to the parent LPS, was found stimulated the formation of the IL-8 in the dose-dependent manner in the human embryonal kidney cells (HEK 293 cells) transfected with TLR4 in combination with MD2.

Animals↗

PubMed2005

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-alpha 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.

Amino Acid Sequence↗

PubMed2005

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.

Acylation↗

PubMed2004

Accumulation of inhibitory kappaB-alpha as a mechanism contributing to the anti-inflammatory effects of surfactant protein-A.

The collectin surfactant protein (SP)-A has been implicated in multiple immunoregulatory functions of innate pulmonary host defense via modulating immune responses both in vitro and in vivo. The aim of the present study was to investigate mechanisms responsible for the anti-inflammatory effects of human (hu) SP-A on the inhibitory kappaB (IkappaB)/nuclear factor (NF)-kappaB signaling pathway in alveolar macrophages (AMs). Initial CD25 expression analysis by flow cytometry of CD14/hu Toll-like receptor 4-transfected Chinese hamster ovary reporter cells demonstrated that SP-A alone does not induce any NF-kappaB-dependent CD25 expression in these cells. In AMs, SP-A pretreatment caused a marked inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation independent of the LPS chemotype used as determined by electrophoretic mobility shift assay. Western blot analysis revealed that SP-A by itself increased the protein expression of IkappaB-alpha, the predominant regulator for rapidly induced NF-kappaB, in a dose- and time-dependent manner without enhancing IkappaB-alpha messenger RNA as determined by reverse transcription-polymerase chain reaction. SP-A did not interfere with LPS-induced serine(32) phosphorylation of IkappaB-alpha but significantly enhanced IkappaB-alpha abundance under LPS-coupled conditions. The data suggest that anti-inflammatory effects of SP-A on LPS-challenged AMs are associated with a SP-A-mediated direct modulation of the IkappaB-alpha turnover in these cells.

Animals↗

PubMed2004

Synthetic lipopeptide adjuvants and Toll-like receptor 2--structure-activity relationships.

Bacterial lipoproteins and their synthetic analogues (sLP) are strong immune modulators of the early host responses after infection. Synthetic lipopeptides are strong adjuvants for the adaptive immune system. Lipoproteins and lipopeptides induce signalling in immune cells through Toll-like receptor-TLR2/TLR1 heterodimers. By screening a combinatorial lipohexapeptide amide collection in an in vitro IL-8 induction assay, we systematically evaluated the potential of 19 proteinogenic amino acids in the peptide moiety of Pam3Cys-lipopeptides to interact with TLR2. New Pam3Cys-lipopeptides with high activity were obtained. Different fatty acids were introduced to investigate the influence of the acyl moiety. Lipopeptides with modifications in the core structure of the unusual amino acid S-glycerylcysteine were synthesized and tested for IL-8 induction via TLR2.

Adjuvants, Immunologic↗