Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model.
The aim of this work was to establish and characterize a cell culture model for lipopolysaccharide (LPS)-induced activation of human endothelial cells. Monocytic THP-1 cells were stimulated for 4 h with 10 ng/ml LPS from Pseudomonas aeruginosa in media containing 10% human plasma. Culture supernatants containing LPS and factors secreted by THP-1 in response to stimulation were applied to human umbilical vein endothelial cells (HUVECs). Nuclear factor-κB (NF-κB) activity, expression of adhesion molecules, and cytokine secretion were quantified. In addition, the effect of adsorptive removal of tumour necrosis factor-α (TNF-α) from the THP-1 culture supernatant on HUVEC activation was assessed. After 4 h of stimulation, THP-1 cells secreted various mediators including TNF-α (854 ± 472 pg/ml), interleukin (IL)-8 (2069 ± 710 pg/ml), IL-18 (305 ± 124 pg/ml), IL-10 (14 ± 5 pg/ml), and IL-1β (24 ± 11 pg/ml). Stimulated HUVECs showed significantly increased NF-κB activity and secreted high amounts of IL-6 and IL-8. Additionally, adhesion molecules ICAM-1 and E-selectin were increased both in the culture supernatant and at the cell surface. Removal of TNF-α from the THP-1 culture supernatant prior to HUVEC stimulation resulted in a decrease in NF-κB activity, expression of adhesion molecules, as well as IL-6 secretion. The cell culture model established in this study permits the monitoring of LPS-induced endothelial activation, which plays a central role in sepsis and may serve to assess the effect of mediator modulation by methods such as extracorporeal blood purification.
Schildberger
Anita
A
Centre for Biomedical Technology, Department for Clinical Medicine and Biotechnology Danube University Krems, Krems, Austria.
Rossmanith
Eva
E
Weber
Viktoria
V
Falkenhagen
Dieter
D
D018929:Cell Culture Techniques; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000276:immunology; D018805:Sepsis / Q000276:immunology* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha