PMN were killed by streptolysin O (SLO) from severe invasive GAS 
PMN death was induced shortly after encounter with severe invasive GAS, and PMN were not in apoptotic death because of low frequency of cells positive for annexin V (data not shown), which was seen in the case of cytolysin-dependent cell injury [15].
GAS produce two cytolysins that may contribute to pathogenesis.
Streptolysin S (SLS) is an oxygen-stable beta-hemolysin and Streptolysin O (SLO) is a pore-forming cholesterol-binding toxin [16].
Therefore, to know the mechanism underlying GAS-mediated killing of PMN, we investigated whether SLO produced by invasive GAS affect survival of PMN in an in vitro migration assay system.
Figure 3A shows that PMN killing by invasive GAS was blocked by anti-SLO Ab in culture medium within lower wells of a transwell system (p=0.018 compared with control Ig), at similar extent by adding free cholesterol in the medium (data not shown).
Furthermore, an SLO deficient mutant from a STSS isolate NIH230 (NIH230slo) lost the killing activity for PMN (Figure 3A), thereby, strongly suggesting that SLO is a key element for PMN killing mediated by invasive GAS.
Contrarily, SLS-deficient mutant from NIH230 strain (NIH230sagA) killed PMN, as efficiently as did parent strain, indicating that SLS is dispensable for killing of PMN mediated by invasive GAS.
SLO and SLS double mutant GAS from NIH230 strain (NIH230slosagA) displayed the killing activity indistinguishable from that of NIH230slo, confirming the primarily role of SLO for GAS-mediated PMN killing.
As shown in Figure 3B, incubation of PMN with supernatant from co-culture of IL-8 and invasive or non-invasive GAS did not affect PMN viability, suggesting that severe invasive GAS causes PMN killing following encounter with bacteria in a contact-dependent manner.
To confirm that SLO activity is increased in invasive GAS strain, we measured SLO hemolytic activity of GAS strains used in this study.
As shown in Figure 3C, SLO activity of severe invasive isolate NIH230 is increased as twice as that of non-invasive strain 1566 (p=0.017).
