Induction of apoptosis in macrophage cell line, J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastasepositive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.