Browsing by Subject "Toll-like receptor"

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Mechanismen der Mastzellaktivierung durch gram-negative Bakterien und Bakterienprodukte aus der Darmflora.
(2006) Krämer, Sigrid; Bischoff, Stephan C.
The role of mast cells (MC) as effector cells in IgE dependent processes like the type 1 allergy has been known for a long time. During the decade, it has been shown that MC are also involved in other pathophysiological processes such as mucosal polyposis, rheumatoid arthritis, inflammatory bowl disease, tissue fibrosis, and atherosclerosis. Furthermore, MC play an important role in the regulation of host defense against microbes, tissue remodeling processes, and neuro-immunology-interaction. The first aim of the present study was to clarify the question whether human intestinal MC express toll-like receptors (TLR), which recognize conserved bacterial and viral components, and can MC be activated through TLR-ligands. The second major focus of the present study was to investigate if the stimulation of human intestinal MC with different E. coli and Shigella strains, respectively, results in an activation of MC and to identify the underlying mechanism(s). Accordingly, human intestinal MC were isolated from surgery tissue with a mechanical and enzymatical protocol. The purity of the MC cultures used in all experiments was between 98 and 100% which was achieved by positive selection (MACS). We could show, that human intestinal MC express mRNA for TLR 1, 2, 3, 4, 5, 6, 8, and 9. However, neither the stimulation with LPS (lipopolysaccharide, TLR 4 ligand), LTA (lipoteichoic acid, TLR 2 ligand), Zymosan (TLR 2 ligand), poly I:C (polyinosinic-polycytidylic acid, TLR 3 ligand), R848 (TLR 7/8 ligand), CpG (C poly G oligo-desoxy-nucleotide, TLR 9 ligand) and non CpG, respectively resulted in a release of histamine, leucotriens, TNF-alpha, or IL-8. Furthermore, mRNA expression levels of TNF-alpha and IL-8 were not induced by any of the treatments. Similar results where found when human intestinal MC were stimulated with E. coli (O101:H-) isolated from human faeces or the probiotic strain E. coli Nissle 1917. Even after stimulation with pathogenic bacteria strains such as the invasive S. flexneri M90T and the fimbriated E. coli, respectively, no induction of any of the parameters mentioned above was found. However, E. coli strains activate the intracellular signal molecule and transcription factors ERK1/2, c-Fos, and AP1, but this activation failed to induce a complete immune answer. In contrast, the hemolytic E. coli stains ATCC 25922 and ATCC 35218 provoked strong activation of intestinal MC. Using the isogenic hemolysin negative E. coli mutants and the hemolysin positive transformants of the probiotic E. coli Nissle 1917 it was shown, that human intestinal MC are sensitive target cells for E. coli alpha hemolysin. Stimulation of MC with sublytic concentration of hemolysin resulted in an induction of TNF-alpha, IL-3, IL-5, IL-6, IL-8 mRNA expression, the release of histamine as well as leucotrien. This activation was found to be regulated by calcium dependent signal cascades. Inhibition of intracellular signal molecules showed that the activation depends on L-typ calcium channels, calcineurin, NFAT and NFkappaB. Prolonged infection with hemolytic E. coli strains resulted in lysis of intestinal MC indicating a biphasic activation of hemolysin.