Innate immune response in human endothelial cells : characterization and regulation of E-selectin, ICAM-I and cytokine expression and the role of Staphylococcus aureus
Sammanfattning: Endothelial cells located on the vessel walls play an important role in inflammation. In response to cytokines or other inflammatory mediators such as microorganisms, endothelial cells express cell adhesion molecules such as E-selectin and ICAM-1 that are involved in binding circulating leukocytes to the endothelium. Since E-selectin is highly glycosylated one aim of this thesis was to investigate the role of N-glycosylation in E-selectin expression and function. Modification of glycosylation was obtained by culturing HUVEC in the presence of soluble glycosylation inhibitors. The result showed that E-selectin is highly glycosylated with N-Iinked, complex type oligosaccharides which were found to be important for the level of cell surface expression and turnover time. However, defect glycosylation did not affect its binding properties as revealed in a static cell-binding assay. Combinations of cytokines can produce a different expression of E-selectin. When TNF-α and IFN-γ were used to stimulate HUVEC an enhanced and prolonged expression of E-selectin was obtained compared to when HUVEC were stimulated with TNF-α alone. This effect could be blocked by monensin, which is an inhibitor of trans-Golgi network processing. This again indicated a role for posttranslational modification in regulation of E-selectin expression.Eighteen clinical isolates of Staphylococcus aureus were analysed for their ability to induce expression of E-selectin and ICAM-1 in HUVEC. Upregulation of adhesion molecules varied between isolates; 17 isolates induced expression of E-selectin and 13 of ICAM-1. Some isolates induced a significant expression of E-selectin without stimulation of ICAM-1, whereas the opposite was not found. Bacterial viability was required for induction of the adhesion molecules. There was no correlation between ability of S. aureus to induce expression of cell adhesion molecules and methicillin susceptibility, pulse field electrophoresis patterns, biochemical characteristics, phage typing or toxin production. Nine cytokines, IL-1ß, TNF-α, IL-6, IL-8, IL-10, IL-12p70, GRO-α, GM-CSF and RANTES, all of importance in the inflammatory process, were analysed in supernatants from HUVEC stimulated with eighteen isolates of S. aureus. All isolates induced IL-6, IL-8, GRO-α, GM-CSF and RANTES. Isolates of S. aureus inducing high expression of one of these cytokines also showed high expression of the other four, indicating a possible common mechanism for regulation of the expression of these cytokines. The ability of individual S. aureus isolates to induce expression of cytokines correlated with their ability to induce expression of ICAM-1 but not E-selectin in HUVEC. No correlation between cytokine profile and S. aureus production of enterotoxin A-D, TSST-1, cytotoxicity, PFGE- and phage pattern or susceptibility to methicillin was observed. Furthermore, culture filtrate from S. aureus induced expression of ICAM-1 and IL-8 in HUVEC. The component(s) from culture filtrates were heat-stable, had a molecular weight of about 100 kDa and the effect was independent of the presence of fetal calf serum in media.
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