Introduction and objectives Pneumolysin (Ply), the major protein virulence factor of the pneumococcus, has been implicated in the pathogenesis of acute lung injury and acute coronary events, both of which are significant causes of mortality, in severe pneumococcal disease. However, the role of Ply in promoting neutrophil/platelet cross-talk, increasingly recognised as a key event in the immunopathogenesis of inflammation-mediated pulmonary and cardiovascular damage is unknown. This issue has been addressed in the current study, which is focused on the effects of exposure of isolated human blood neutrophils to Ply on the production of the platelet-targeted, pro-inflammatory lipids, platelet-activating factor (PAF) and thromboxane A2 (TXA2).
Methods Neutrophils, isolated from the blood of healthy, adult humans, were suspended at a concentration of 2 × 106/ml in Hanks balanced salt solution and preincubated for 10 min at 37°C followed by addition of recombinant Ply (5–80 ng/ml), or the pneumolysoid, delta6Ply (80 ng/ml, negative control), or the calcium ionophore, A23187, (2 μM, positive control). After 5 min of incubation, the reactions were terminated and PAF and TXA2 assayed in the cell-free supernatants using sandwich ELISA procedures.
Results These are shown in the accompanying Table 1. Exposure of neutrophils to Ply resulted in dose-related enhancement of production of PAF, which achieved statistical significance at concentrations ≥20 ng/ml of the toxin, while delta6Ply was ineffective, and A23187 extremely potent. Similar, but less impressive effects were noted in the case of TXA2.
Conclusion Ply, via its pore-forming activity, activates the production of PAF and, to a lesser extent, TXA2, by neutrophils, potentially augmenting pro-inflammatory cross-talk between these cells and platelets, an activity of the toxin which may contribute to the immunopathogenesis of lung and cardiac injury in severe pneumococcal disease.
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