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Innate immune response in human endothelial cells: characterization and regulation of E-selectin, ICAM-I and cytokine expression and the role of Staphylococcus aureus
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 52 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 804
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26669Local ID: 11235ISBN: 91-7373-493-4 (print)OAI: oai:DiVA.org:liu-26669DiVA: diva2:247218
Public defence
2003-09-26, Elsa Brändströmsalen, Hälsouniversitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-16Bibliographically approved
List of papers
1. Role of N-linked glycosylation in expression of E-selectin on human endothelial cells
Open this publication in new window or tab >>Role of N-linked glycosylation in expression of E-selectin on human endothelial cells
1995 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 25, no 9, 2452-2459 p.Article in journal (Refereed) Published
Abstract [en]

E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84628 (URN)10.1002/eji.1830250907 (DOI)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
2. Interferon-γ enhancement of E-selectin expression on endothelial cells is inhibited by monensin
Open this publication in new window or tab >>Interferon-γ enhancement of E-selectin expression on endothelial cells is inhibited by monensin
1997 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 46, no 4, 338-343 p.Article in journal (Refereed) Published
Abstract [en]

The expression of E-selectin reaches a maximum 4–6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-α (TNF-α) and then declines to basal level within 24 h. If interferon-γ (IFN-γ) is added to the cell culture medium together with TNF-α the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-γ induced persistent surface expression of E-selectin. SDS–PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-γ produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-γ/TNF-α compared to TNF-α alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monensin, a potent inhibitor of late Golgi function, together with both TNF-α and IFN-γ, the additive effect of IFN-γ on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-γ induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-γ/TNF-α in the presence of several different inhibitors of N-glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84629 (URN)10.1046/j.1365-3083.1997.d01-135.x (DOI)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
3. Variations among clinical isolates of Staphylococcus aureus to induce expression of E-selectin and ICAM-1 in human endothelial cells
Open this publication in new window or tab >>Variations among clinical isolates of Staphylococcus aureus to induce expression of E-selectin and ICAM-1 in human endothelial cells
2002 (English)In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 32, no 3, 227-235 p.Article in journal (Refereed) Published
Abstract [en]

Eighteen clinical isolates of Staphylococcus aureus, nine methicillin-sensitive and nine methicillin-resistant, were investigated for their ability to induce expression of E-selectin and ICAM-1 in human endothelial cells. 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. The kinetics of ICAM-1 expression in S. aureus-infected cells differed from those stimulated with interleukin-1β (IL-1β). On the other hand, expression of E-selectin was very similar in S. aureus-infected and IL-1β-stimulated cells. There was no correlation between ability of S. aureus to induce expression of cell adhesion molecules, methicillin susceptibility, pulse field gel electrophoresis patterns, biochemical characteristics, phage typing and toxin production.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26508 (URN)10.1016/S0928-8244(01)00306-6 (DOI)11065 (Local ID)11065 (Archive number)11065 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Cytokine expression in human endothelial cells stimulated with clinical isolates and culture filtrate of Staphylococcus aureus
Open this publication in new window or tab >>Cytokine expression in human endothelial cells stimulated with clinical isolates and culture filtrate of Staphylococcus aureus
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Human umbilical vein endothelial cells (HUVEC) were infected for 24 h with 18 well-characterized, noninvasive Staphylococcus aureus isolates and the supernatants were analyzed for IL-1ß, TNF-α, IL-6, IL-8, IL-10, IL-12p70, GRO-α, GM-CSF and RANTES by immuno assay. All isolates induced expression of IL-6, IL-8, GRO-α, GM-CSF, and RANTES. The highest concentrations were observed for IL-6, IL-8 and GRO-α, which reached levels close to that of HUVEC stimulated with LPS (0.1µg ml-1). The magnitude of cytokine expression varied between isolates. S. aureus inducing high expression of one of these cytokines also showed simultaneous high expression of the other four, indicating a common ability of individual S. aureus to induce high or low expression of these cytokines. IL-1ß, TNF-α, IL-10 and IL-12p70 were not upregulated by any of the S. aureus isolates. No correlation between cytokine profile and S. aureus production of enterotoxin A-D, TSST-1, cytotoxicity, PFGE and phage pattem or susceptibility to methicillin was observed. The ability of individual S. aureus to induce expression of cytokines correlated with their ability to upregulate ICAM-1, but not E-selectin, in HUVEC. Similarly, a heat-stable, high molecular weight component(s) from sterile culture filtrate of S. aureus upregulated expression of IL-8 and ICAM-1 in HUVEC.

Our results show that individual clinical isolates of S. aureus vary in ability to directly stimulate human endothelial cells to upregulate cytoldnes that promote leukocyte recruitment and inflammation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84631 (URN)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2012-10-16Bibliographically approved

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