RESUMO
Neutrophils isolated from human peripheral blood added to a monolayer of human endothelial cells (ECV-304 cell line) stimulated with LPS (100 ng ml(-1)) resulted in: (a) neutrophil activation, measured by spreading and release of leukotriene B(4) (LTB(4)); (b) neutrophil degranulation, measured by release of matrix pro-metalloproteinase-9 (proMMP-9) and (c) loss of the monolayer integrity due to detachment of the endothelial cells. Stimulation of endothelial cells with tumor necrosis factor-alpha (TNF-alpha 10 ng ml(-1)) or interleukin-1 (IL-1; 10 ng ml(-1)) induced a similar dose-dependent increase in the neutrophil activation and endothelial cell detachment. Pre-treatment of LPS-activated ECV-304 cells with [Phe22]BigET-1(19-37) (10(-9) M; an inhibitor of endothelin converting enzyme (ECE)) or addition of BQ-123 (10(-6) M; a selective endothelin A (ET(A)) receptor antagonist) to the co-cultures, significantly reduced neutrophil spreading (50-70% inhibition) as well as the levels of LTB(4) (70-100% inhibition) and proMMP-9 (40-50% inhibition) in the co-culture supernatants. In addition, the detachment of endothelial cells was also reduced (60-75% inhibition). Moreover, the exogenous addition of ET-1 (10(-9) M) to neutrophil suspensions induced neutrophil spreading and release of LTB(4) and proMMP-9. Taken together, these findings indicate that neutrophils added to stimulated endothelial cells in the co-culture system employed in this study, get activated by products of these cells and degranulate. In parallel, the detachment of endothelial cell monolayer from the culture plates, possibly by the action of neutrophil granule-derived gelatinases, is observed. Endothelins (ETs) produced by the endothelial cells are suggested to play an essential role in these phenomena.
