The activation of caspase-3 and DNA fragmentation in B cells phagocytosed by macrophages

 Apoptotic signaling of mammalian cells involves two pathways: the death receptor and mitochondrial pathways. In this in vivo study, we investigated apoptotic signaling of B cells in mouse germinal centers (GCs) of gut-associated lymphoid tissues (GALTs) using transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), immunofluorescence of members of caspase family and cFLIPL, and caspase activity assay. It was very difficult to ultrastructurally differentiate B cells undergoing apoptosis from B cells differentiating into memory cells or plasma cells among B cells constituting GCs. Isolated B cells in GCs showed no active form of caspase-3 or TUNEL immunoreactivity, but expressed cFLIPL. Contrary to isolated B cells, apoptotic B cells phagocytosed by macrophages exhibited immunoreactivity of the active form of caspase-3 and TUNEL, but lacked the cFLIPL expression. The caspase activity assay in GALTs clearly showed intense activity of caspase-3, caspase-9, and caspace-8 that was high in order. Therefore, the death receptor pathway accompanying the increased activity of caspase-3 and caspase-8 may be blocked by the expression of cFLIPL in B cells of GALTs. Moreover, both the activation of caspase-3 and DNA fragmentation first occur only when B cells are phagocytosed by macrophages.