CONCANAVALIN A-INDUCED APOPTOSIS IN MURINE MACROPHAGES THROUGH A CA2-INDEPENDENT PATHWAY()

Concanavalin A (ConA), normally a mitogen of T lymphocytes, was found to induce apoptosis or programmed cell death in murine peritoneal macr ophages. The following observations support this assertion: 1) incubat ion of peritoneal macrophages or cultured PU5-1.8 macrophage cells wit h ConA caused a dose- and time-dependent reduction of mitochondrial de hydrogenase activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, 2) treatment of cells with Co nA induced formation of apoptotic bodies as seen under the confocal la ser scanning microscope, 3) challenge of cells with ConA produced a co nsiderable amount of cell debris with DNA content next to G0 phase as revealed by flow cytometry and 4) ConA was able to elicit DNA fragment ation in these cells, The involvement of Ca2+ in mediating the apoptos is was studied in single cells by confocal laser scanning microscope u sing the Ca2+ fluorescence dye, fluo-3. Our results show that ConA ind uced an immediate rise of intracellular free Ca2+ concentration as wel l as opening of Ca2+ channels on cell surface. But when the cells were treated with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic aci d/AM (BAPTA/AM), a Ca2+ chelator, to buffer the rise of internal Ca2+, ConA still caused DNA fragmentation, Furthermore, injection of Ca2+ i nto the cell with ionomycin had no stimulatory effect on DNA fragmenta tion. These results suggest that Ca2+ changes induced by ConA are not a prerequisite for apoptosis in macrophages.