Induction of necrosis and apoptosis of neutrophil granulocytes by Streptococcus pneumoniae

Abstract

Apoptosis followed by macrophage phagocytosis is the principal mechanism by which neutrophil granulocytes (PMN) are removed from the site of inflammation. To investigate whether Streptococcus pneumoniae causes apoptosis of PMN, we exposed PMN to viable and heat-killed pneumococci and purified pneumococcal cell walls (PCW). The occurrence of PMN cell death was quantified by flow cytometry using annexin V/propidium iodide labelling of the cells. Intracellular histone-associated DNA fragments were quantified by ELISA. The presence of apoptosis was confirmed by in situ tailing. Exposure of PMN to viable pneumococci caused necrosis of the cells. The pneumococcal cytotoxin pneumolysin, the bacterial production of hydrogen peroxide, and PCW contributed to necrosis. Heat-killed pneumococci accelerated the process of apoptosis observed in cultivated non-stimulated PMN in vitro. These results demonstrated that pneumococci induce PMN cell death. Depending on the intensity of the stimulus, PMN necrosis and apoptosis were observed.

Fig. 1

FACS analysis of PMN after labelling the cells with annexin V–FITC and propidium iodide (PI). Viable cells were negative for annexin V–FITC and PI (lower left quadrant). Annexin V labelling indicated apoptosis (lower right quadrant). Cells which were stained with PI were necrotic (upper quadrants) [32]. Unstimulated freshly isolated PMN (a) and PMN after 19 h in vitro culture: unstimulated controls (b), after addition of 10⁷ viable pneumococci (D39)/ml (c), 10⁷ viable pneumolysin-deficient pneumococci (D39ply::pJDC9)/ml (d), 10⁷ viable pneumolysin-deficient pneumococci (D39ply::pJDC9)/ml + catalase (2000 EU/ml) (e), 10⁷ heat-killed pneumococci (D39)/ml (f).

Fig. 2

Apoptosis was determined by ELISA measuring the concentration of cellular histone-associated DNA fragments. The concentrations were determined 0 h, 2 h, and 4 h after the addition of heat-killed pneumococci (equalling 10⁷ colony-forming units (CFU)/ml) or pneumococcal cell walls (PCW; 50 μg/ml) to the in vitro culture of PMN. OD, Optical density.

Fig. 3

Detection of apoptotic nuclei (dark staining) of PMN by in situ tailing. The tailing reaction was performed 24 h after PMN had been isolated. Controls rarely showed apoptosis (A), whereas in cultures stimulated with heat-killed pneumococci a high rate of PMN apoptosis was observed (B).