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-kappa B (NF-kappa B) activity, expression of adhesion molecules, and cytokine secretion were quantified. In addition, the effect of adsorptive removal of tumour necrosis factor-alpha (TNF-alpha) from the THP-1 culture supernatant on HUVEC activation was assessed. After 4 h of stimulation, THP-1 cells secreted various mediators including TNF-alpha (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-1 beta (24 +/- 11 pg/ml). Stimulated HUVECs showed significantly increased NF-kappa B 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-alpha from the THP-1 culture supernatant prior to HUVEC stimulation resulted in a decrease in NF-kappa B 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.