Death of germinal center B cells without DNA fragmentation

Abstract

During the selection of B cells within germinal centers (GC) on the basis of their affinity for T-dependent antigen, B cells not positively selected are eliminated within GC. This process of B cell death has been considered to be apoptosis. In a recent study, we have reported that, although a substantial number of thymocytes were considered to be dead because of their extremely small cell size and heavy chromatin condensation even though they were not yet phagocytosed (pyknosis), they were devoid of DNA fragmentation, the most characteristic feature for apoptosis. In this study, we examined in vivo the mechanism of B cell death within GC by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA double-strand breaks. TUNEL+ B cells were scattered throughout the upper dark and the light zones of GC. Double staining of the sections by the TUNEL method and acid phosphatase (AcP) activities showed that all the TUNEL+ B cells were phagocytosed by macrophages. Light microscopic and ultrastructural studies revealed the presence of small unphagocytosed B cells within the light zone. These cells are undoubtedly dead because they were much smaller than surrounding lymphoid cells and have a heavy chromatin condensation. Furthermore, ultrastructural detection of DNA fragmentation confirmed that these small unphagocytosed B cells were TUNEL-, implying that DNA fragmentation is not primarily involved in the cell death process of these small dead B cells. These results indicate that most B cells, not positively selected and thus destined to be eliminated, die within GC without DNA fragmentation, and are subsequently phagocytosed by macrophages and become TUNEL+. Typical apoptosis, characterized by DNA fragmentation in situ, is not the predominant type of cell death that occurs during the selection of B cells in GC.

IgH V-Region Sequence Does Not Predict the Survival Fate of Human Germinal Center B Cells

“…This is comparable to levels reported by Brieva and colleagues (43) and Groot and colleagues (21). Earlier investigations show that DNA degradation and caspase 3 activation generally do not occur until after B cells are engulfed by macrophages (58,59), therefore rendering TUNEL assays and use of fluorometric caspase substrates less reliable for isolating early apoptotic, nonphagocytized GC B cells. Consistent with these reports, our efforts to sort intact GC B cells based on simultaneous staining of AnV and caspase 3 (active form) proved to be problematic (data not presented).…”

Glucocorticoid-Induced Thymocyte Death in the Murine Thymus: The Effect at Later Stages.

“…Thymocytes die of neglect by pyknosis. We also demonstrated by the TUNEL method that B lymphocytes in the germinal center die without DNA fragmentation, during the affinity maturation (NAKAMURA et al, 1996). Here we reported that, even in the glucocorticoid treated thymus, most thymocytes die by pyknosis without showing DNA fragmentation, contrary to what has been postulated about the glucocorticoid-induced thymocyte death (WYLLIE, 1980;COHEN and DUKE, 1984;TANUMA and SHIOKAWA, 1994).…”

“…10a) (NAKAMURA et al, 1996). By this point, small pyknotic thymocytes probably induced by glucocorticoid treatment were also observed predominantly in the cortex.…”

“…We previously showed that DNA fragmentation was not the primary event in glucocorticoid-induced thymocyte death in vivo within 4h after glucocorticoid treatment (NAKAMURA et al, 1996). In the present paper, we performed a kinetic study to investigate the late effect of glucocorticoid on thymocyte death in vivo.…”

The Relationship between Apoptosis and Keratinization in Human Epidermis.

“…In contrast to the above-mentioned findings, recent studies on the death of germinal center B cells [14] and glucocorticoid-induced thymocyte death [7] show that the small dead B cells and thymocytes characterized by nuclei with heavy chromatin condensation were ISEL-negative and DNA fragmentation is not primarily involved in the cell death. Therefore, DNA fragmentation detected by ISEL is independent of chromatin condensation.…”