Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model.

TLDR: 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.

Abstract: 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