Induction of Apoptosis in Macrophage Cell Line, J774, by the Cell‐Free Supernatant from Pseudomonas aeruginosa

Abstract

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 (elastase‐positive 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.

Survival of Virulent Mycobacterium tuberculosis Involves Preventing Apoptosis Induced by Bcl‐2 Upregulation and Release Resulting from Necrosis in J774 Macrophages

“…For some experiments, the cells were cultured in culture dishes containing glass slides. Following three washes with phosphate-buffered saline (PBS), they were cultured in fresh medium before use in the various assays (27).…”

“…M. tuberculosis was killed at 56 C for 1 hr (14), after which the supernatants were harvested by centrifugation at 2,000ϫg for 15 min (OD 570 ϭ1.80), obtained by filtration through a 0.22-µm-pore-size membrane filter (Millipore Corporation, Bedford, Mass., U.S.A.). After this, the bacilli were stored at Ϫ20 C before use (27).…”

“…The viabilities of the cells were assessed at different times by the trypan blue dye exclusion method. Three hundred cells on each slide were counted and every experiment was performed in triplicate (27).…”

“…Ind.). The cells were then stained with Hoechst 33258 (Sigma-Aldrich Corp.) as previously described and the nuclei were observed under a fluorescent microscope (27). Other slides were permeated with 0.5% Triton X-100 for 2 min at room temperature.…”

“…Other slides were permeated with 0.5% Triton X-100 for 2 min at room temperature. The samples were labeled using an MEBSTAIN Apoptosis Kit II (Medical and Biological Laboratories, Nagoya, Japan) for in situ cell death detection according to the manufacturer's protocol and subsequently observed under a fluorescent microscope (27).…”