Proinflammatory Interactions of Pneumolysin with Human Neutrophils

Abstract

The effects of pneumolysin on the proinflammatory activity of human neutrophils, as well as on cation fluxes in these cells, have been investigated. Superoxide production, release of elastase, CR3 expression, phospholipase A2 activity, and alterations in membrane potential were measured by use of lucigenin-enhanced chemiluminescence and colorimetric, flow cytometric, radiometric, and spectrofluorimetric procedures, respectively; and cation fluxes were measured by use of 45Ca2+ and 86Rb+ and by fura-2 spectrofluorometry. Pneumolysin at concentrations >1.67 ng/mL caused influx of Ca2+ and increased phospholipase A2 activity and CR3 expression, which were associated with enhanced superoxide production and release of elastase after activation of the cells with the chemotactic tripeptide FMLP. At the same concentrations, pneumolysin caused efflux of K+ and membrane depolarization. The effects of pneumolysin on cation fluxes were not attributable to inhibition of Ca2+-adenosine triphosphatase (ATPase) or Na+, K+-ATPase. Pneumolysin potentiates the proinflammatory activities of neutrophils by a pore-forming mechanism resulting in Ca2+ influx.

Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

“…17,18 These pro-inflammatory interactions of the toxin with neutrophils were associated with influx of Ca 2+ , 11 an event which precedes and is a pre-requisite for mobilization of secondary/tertiary granules 19 and eicosanoid production. 20 Although Δ6 PLY at 1000 ng/ml caused modest Ca 2+ influx following a protracted lag phase, the resultant increment in cytosolic Ca 2+ did not achieve the necessary threshold for either degranulation or production of LTB 4 .…”

“…11 The neutrophils, which were routinely of high purity (>90%) and viability (>95%), were resuspended to 1x10 7 cells/ml in phosphate-buffered saline (PBS, 0.15 M; pH 7.0).…”

“…11 Briefly, neutrophils (10 7 /ml in PBS) were loaded with fura-2/AM (2 mM) for 30 min at 37°C after which they were washed and resuspended in Hanks' Balanced Salt Solution (HBSS, pH 7.4, indicator-free), and preincubated for 5 min at a concentration of 2x10 6 /ml in HBSS. The cells were then transferred to disposable reaction cuvettes that were maintained at 37°C in a thermoregulated Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths set at 340 and 500 nm respectively.…”

“…Addition of WT/PLY (8 ng/ml) to neutrophils was followed in succession by a short lag phase (~20sec) and a steep increase in fluorescence intensity due to influx of extracellular Ca 2+ , 11 which rapidly reached a sustained plateau phase. Δ6 PLY, at concentrations of 8 and 40 ng/ml, did not promote Ca 2+ influx, while at 1000 ng/ml the mutant toxin caused a slight influx of Ca 2+ following a prolonged lag phase of several minutes.…”

“…10 Critical criteria for further development include immunological and histological evaluation of protective efficacy and safety in animal models, as well as in vitro determination of attenuation of pore-forming activity in haemolytic assays. This latter assessment, although well-accepted, does not, however, detect the sub-lytic activity which underpins the potentially harmful pro-inflammatory activity of the toxin with other cell-types such as neutrophils 11 and epithelial cells. 12 In the current study, we have therefore compared the effects of WT/PLY and Δ6 PLY on several Ca 2+ -dependent pro-inflammatory activities of human neutrophils in vitro.…”