Study of the mechanisms of killing of Mycobacterium bovis BCG by apoptosis in J774 murine macrophages.
OBJECTIVE
To determine the relationship between the mechanism of apoptosis and intracellular killing of Mycobacterium bovis BCG. Apoptosis, "programmed cell death"--a physiologically beneficial and distinct form of cell death.


METHODS
In vitro study was carried out in murine macrophage cell line J774 that was infected with Mycobacterium bovis BCG in different set of conditions. Percentage of surviving BCG and apoptotic cells was determined.


METHODS
IFN-g and/or LPS-activated and non-activated J774 mouse macrophage cells were infected with BCG in a ratio of 1:5. The morphology of the host cells was studied after 4 hours, 24 hours and 48 hours of infection in cytospins stained with Jenner-Giemsa. Surviving bacteria were counted by incorporation of radiolabelled-uridine after cell lysis.


RESULTS
Both in the activated and non-activted J774 cultures some cells undergo apoptosis. In cells activated with IFN-g or LPS without BCG, less than 10% of cells were found to be apoptotic. More apoptosis was seen when LPS-activated cells were infected with BCG. In the cells activated with IFN-g or LPS-activated cells the percentage of apoptotic cells was much higher than in non-activated cells or cells activated with either INF-g or LPS alone. After 24 hours culture, without BCG, about 15% of the cells were found to be apoptotic and with BCG infection this increased to 23% (p < 0.01). The level further increased after 48 hours of infection. BCG growth inhibition was observed in both non-activated J774 cells and cells activated with LPS, INF-g or both and was sustained to 48 hours of co-culture.


CONCLUSIONS
It is evident that BCG-infected J774 cells undergo apoptosis in the presence of a high concentration of RNI and/or ROI. During this process the cells shrink considerably in volume with the removal of water that may concentrate toxic products in the cell. The increased concentration of toxic species and the disorganisation of the phagocytic vacuoles may account for the enhanced stasis and/or death of the intracellular micro-organisms. We conclude that host cell apoptosis may arrest the growth and account for the death of the intracellular mycobacterial pathogen.
Ahmad
A
A
Department of Microbiology, University of Karachi.
Khan
M
M
Raykundalia
C
C
Catty
D
D
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D009163:Mycobacterium bovis