[Human lung epithelial cells produce interleukin-8 through protease-activated receptor 1]

To investigate the actions of protease-activated receptor 1 (PAR1) agonists and thrombin on the secretion of interleukin-8 (IL-8) from human lung epithelial cells.

A549 cells were cultured in a 12-well culture plate. The challenge was performed by addition of various concentrations of PAR1 agonist peptides SFLLR and its reverse peptides RLLFS, thrombin or hirudin, a thrombin inhibitor, into each well, respectively. After 2 or 16 h, the reactions were terminated by removal of the supernatant from each well. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-8 in the supernatants.

Following a 16-hour incubation, SFLLR was able to induce concentration-dependent secretion of IL-8. The maximum release of IL-8 was increased nearly 16 fold more than the baseline release. The reverse PAR1 agonists had little effects on IL-8 release. Thrombin was also able to induce concentration- dependent secretion of IL-8. As low as 1 kU/L thrombin was able to induce IL-8 release from the epithelial cells, and the maximum accumulated release of IL-8 was observed with 10 kU/L thrombin, which was 7.5 fold the baseline release. Thrombin inhibitor hirudin could inhibit thrombin-induced secretion of IL-8. The time course showed that the actions of PAR1 agonist peptides SFLLR and thrombin initiated at 2 h and reached the peak at 16 h.

PAR1 agonist peptides and thrombin are potent secretogogues of IL-8 release from cultured human lung epithelial cells, and PAR1 antagonists and thrombin inhibitor may possess the ability to inhibit airway inflammation.