Human endothelial cells are activated by interferon-gamma plus tumour necrosis factor-alpha 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 NG-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.

Comparative analysis of three methods to assess viability of mammalian cells in culture.

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3% sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays.

Comparative analysis of three methods to assess viability of mammalian cells in culture.

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3

sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays.