We have previously shown that IL-8 is the major chemoattractant present in monocyte supernatants after 24h incubation with GBS. IL-8 release is first detectable at 4h and continues throughout the period of stimulation. The current work was undertaken to help determine triggers for GBS-induced release of IL-8 by monocytes. Studies by others have indicated that phagocytosis enhances IL-8 release; we hypothesized that phagocytosis of GBS would similarly enhance IL-8 production, and that soluble factors present in conditioned media would be sufficient to induce IL-8. Peripheral blood monocytes from adult donors were separated by density centrifugation and allowed to adhere to plastic in serum-free media (106 cells/ml). Formalin-inactivated, unopsonized type III GBS were added (108 CFU/ml) to some wells while others were incubated with media alone. At 4h, supernatants were removed from the GBS-stimulated wells, filtered to remove bacteria and layered over cells which had previously been incubated with media alone. These monocytes were further incubated with this conditioned media for 20h (total, 24h). Supernatants were harvested, filtered and frozen at-70°C. IL-8 was assayed (n=3 expts) by ELISA (R&D Systems):Table Thus, incubation with conditioned media induced significantly more IL-8 than if the monocytes had been incubated with GBS for the entire 24h. Supernatants also induced IL-8 if they were overlayed at 0.5h(29.1 ± 9.3 ng/ml) or 2h (82.4 ± 22.8 ng/ml). These data support two conclusions: first, phagocytosis of GBS is not required for IL-8 release by monocytes. A soluble factor, either of bacterial or host origin, seems sufficient to induce IL-8 production. Secondly, continued contact with GBS diminishes the IL-8 response. Further studies will evaluate the extent to which this effect may hamper neutrophil recruitment to the site of invasion, enhancing risk of overwhelming infection.

Table 1
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