Abstract

Endothelial cells play roles in regulating blood flow and in control of inflammation, both of which play important roles in atherogenesis. It is known that the NLRP3 inflammasome is involved in innate defence mechanisms in monocytes and macrophages and requires two steps for activation, signal 1 and signal 2, which result in the production of active IL-1β and IL-18. The aim of this study was to investigate NLRP3 inflammasome activation in human endothelial cells. Phorbol 12-myristate 13-acetate (PMA; 5 ng/ml) differentiated THP-1 cells (positive control), human umbilical vein endothelial cells (HUVEC) and EA.hy926 cells were ‘primed’ for signal 1 with lipopolysaccharide (LPS) (0.1–5 µg/ml) for 24 hours and then activated (signal 2) by exposure to ATP (300 µM) for 1 hour. Using Western blotting, NLRP3 protein, pro-IL-1β, pro-IL-18, active IL-1β and active IL-18 were observed in THP-1 cells demonstrating priming and activation of the inflammasome as expected. EA.hy926 cells but not HUVEC expressed pro-IL-1β. Inflammasome activation was confirmed by ELISA which detected any active end products in the cell culture supernatant. THP-1 cells secreted IL-1β and IL-18 as expected. However, for endothelial cells, only EA.hy926 cells showed some low level expression of active IL-1β. In conclusion, although there was evidence of NLRP3 inflammasome priming for both endothelial cell types, only EA.hy926 cells showed extracellular IL-1β production. Other end products of NLRP3 inflammasome activation may be worthy of investigation in order to fully describe the response of endothelial cells to innate stimuli.