Human endothelial cells are activated by interferon-γ plus tumour necrosis factor-α to kill intracellular Pseudomonas aeruginosa

Proinflammatory cytokines have been shown to activate endothelial cells. To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha) for 24 hr and exposed to P. aeruginosa suspension for 1 hr. Light microscopy showed that activated cells internalized significantly more bacteria than control cells. To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay. In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection. In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower. Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants. HUVEC anti-P. aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the L-arginine analogues aminoguanidine and N-G-monomethyl-L-arginine (L-NMMA), but was significantly inhibited by catalase. Our results indicate that HUVEC can be activated by IFN-gamma plus TNF-alpha to kill IC P. aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity.