Objectives: To investigate the susceptibility and responses of human brain target cells to lethal and sublethal ILY concentrations.
Methods: Primary brain endothelial cells (HBMEC), astrocytes (HA) and a neuronal cell line (DAOY) were treated for 1h or 2h with purified ILY (0.000125 - 0.1µg/ml). Cell viability, damage and recovery were assessed using MTT assay, LDH release and AlamarBlue™. Cellular response assays included cytokine release (IL-1α , IL-1β, TNF-α and IL-8) by ELISA, cell cycle quantification and caspase activity by flowcytometry and NF-κB p65 subunit nuclear translocation plus cytoskeletal changes by immuno-fluorescence. ILY-exposed HBMEC supernatants were used in a neutrophil migration assay.
Results: Dose dependant cell viability decrease was observed with all target cells. ILY susceptibility varied between cell types, with astrocytes and neuronal cells 10-fold more susceptible than brain endothelial cells. Maximal IL-8 release was at lowest (0.000125µg/ml) ILY concentration (3000pg/ml from HBMEC). Increasing IL-1α release was directly ILY dose dependant whereas IL-1β and TNF-α release was negligible. Alteration of the cytoskeleton accompanied by NF-κB nuclear translocation was observed. Dose dependant activation of caspases in an apoptotic cell population with Sub-G1 arrest of cells was evident. HBMEC supernatants also caused migration of neutrophils across an endothelial membrane barrier.
Conclusions: These in-vitro data support the hypothesis that sublethal ILY levels may play an important role in brain abscess pathogenesis. NF-κB activation in brain endothelial cells and astrocytes (blood-brain barrier) may cause release of pro-inflammatory cytokines, especially IL-8, and regulation of the immune response at foci of infection in-vivo. Cell death during infection may not result simply as a direct consequence of pore formation on target membranes but also by activation of the apoptotic caspase cascade.