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

^* To whom correspondence should be addressed. E-mail: viktoria.weber{at}donau-uni.ac.at.

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

First published on August 26, 2009
Innate Immunity 2009, doi:10.1177/1753425909341885


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