Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood

OBJECTIVE: To investigate the role of endotoxin-induced inflammatory mediators in blood on the permeability of endothelial monolayers. DESIGN: Whole blood of healthy volunteers was treated with bacterial lipopolysaccharide (Escherichia coli, B55:05), and the resultant plasma was added to human umbilical venular endothelial cells (HUVEC) cultured on semipermeable membrane inserts (Transwells). SETTING: University hospital laboratory. SUBJECTS: Whole blood of healthy volunteers. INTERVENTIONS: Donor plasma was treated with excess antibodies against either tumor necrosis factor-alpha, interleukin-1beta, or both, before the incubation on HUVEC. MEASUREMENTS AND MAIN RESULTS: The permeability of HUVEC monolayers to fluorescent-labeled albumin and dextran was measured over a 6-hr period, after removal of the stimulus. The production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated whole blood was determined by radioimmunoassay. Individually, lipopolysaccharide (10 microg/mL), tumor necrosis factor-alpha (10 ng/mL), and interleukin-1beta (50 ng/mL) all increased endothelial permeability by about 2.5-fold. A much larger increase could be achieved by preincubation of lipopolysaccharide (10 microg/mL) in whole blood: the resultant plasma induced a ten-fold increase of the permeability. The permeability response after preincubation of lipopolysaccharide in whole blood was time- and dose-dependent. Moreover, this treatment increased the sensitivity of endothelial monolayers to lipopolysaccharide by a factor of several thousand-fold: Whereas high doses of lipopolysaccharide were required for direct stimulation of the permeability, picomolar amounts of lipopolysaccharide in whole blood induced a similar increase. Significant amounts of tumor necrosis factor-alpha and interleukin-1beta were produced in blood at similar doses of lipopolysaccharide. The addition of antibodies against tumor necrosis factor-alpha or interleukin-1beta to plasma partially but significantly abrogated the permeability increase. However, a complete inhibition could be achieved by the simultaneous addition of anti-tumor necrosis factor-alpha and anti-interleukin-1beta to plasma. CONCLUSIONS: Although lipopolysaccharide is capable of directly inducing endothelial permeability, blood-borne tumor necrosis factor-alpha and interleukin-1beta mediate lipopolysaccharide-induced endothelial permeability at low endotoxin concentrations. These findings support the idea that multifactorial inhibition of inflammatory mediators may improve survival in septic patients.