Pneumococci and PCW trigger PCD in BMECs. (A and C) Unchallenged BMECs. Living pneumococci (R6, 10⁷ CFU/ml, 12 hours) induced the appearance of TUNEL-positive BMECs (B) and shrinkage and condensation of the nuclei by ethidium bromide/acridine orange staining (D). (E) BMECs incubated with PCW (10⁷ CFU equivalents, 72 hours) underwent shrinkage, condensation, and fragmentation of the nuclei by ethidium bromide/acridine orange staining. Arrows indicate apoptotic bodies. (F) Pneumococci (D39) induced nuclear fragmentation (arrow) in endothelial cells of the vessel wall of capillaries in experimental mouse meningitis. (G) Electron microscopy showed a normal nucleus in the control culture. (H) Shrinkage and condensation of the nucleus occurred after challenge with living pneumococci (R6, 10⁷ CFU/ml, 4 hours). (I) Nuclear fragmentation characterized PCD by PCW (10⁷ CFU equivalents, 72 hours). Scale bars: 10 μm (A–F) and 1 μm (G–I). (J) Pneumococci (D39) caused dose- and time-dependent PCD in BMECs. No BMECs survived 18 hours after pneumococcal challenge. Co, control; n.d., not done. (K) PCW triggered a dose- and time-dependent protracted PCD. (L) The absence of 1 toxin, either pneumolysin (plnA^–) or H₂O₂ (spxB^–), did not prevent PCD compared with wild-type D39. Absence of both toxins significantly decreased PCD. Addition of catalase (Cat; 1,250 U/ml) to plnA^– resulted in only a minor enhancement of protection of BMECs compared with plnA^–spxB^– after 12 hours. All data are presented as mean ± SD. *P < 0.05 (ANOVA and Student-Newman-Keuls test).