No abstract

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.

OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators.

SummaryThe aim of this study was to elucidate the time course of the permeability response of endothelial monolayers after exposure to plasma obtained from lipopolysaccharide (LPS)-treated human whole blood; to investigate the role of apoptosis in monolayer permeability, and to inhibit the permeability increase, particularly after addition of the plasma stimulus. Human umbilical vein endothelial cells (HUVEC) were cultured on semiporous membranes and the permeability for albumin was measured after exposure, according to different schedules, to LPS-conditioned plasma. Apoptotic HUVEC were measured by both flow cytometry and ELISA. A variety of agents, including antibodies against cytokines, inhibitors of NF-κ κ κ κ B, and a caspase inhibitor, were added to HUVEC, either prior to or after the stimulus. A maximum increase of the permeability was achieved after 4-6 h of exposure to LPSconditioned plasma. This response was not accompanied by an increase in the number of apoptotic HUVEC. Administration of antibodies against both Tumour Necrosis Factor-α α α α (TNF-α α α α ) and Interleukin-1 β β β β (IL-1 β β β β ) to HUVEC within 1 h after stimulation significantly reduced the permeability increase. Similarly, pyrollidine di-thiocarbamate (PDTC), but not N-acetylcysteine, could prevent the permeability response, and was still effective when added within 2 h after LPS-conditioned plasma. The TNF-α α α α /IL-1 β β β β signal present in LPS-conditioned plasma appears to increase endothelial permeability through intracellular pathways that very likely involve the activation of NF-κ κ κ κ B. Although poststimulatory inhibition of the permeability response proves to be possible with agents such as PDTC, the window of opportunity appears very small if placed in a clinical perspective.

The relative contribution of the pro-inflammatory cytokines tumour necrosis factor (TNF)-a and interleukin (IL)-1b and the lipopolysaccharide (LPS)-induced pathways that result in endothelial activation during sepsis are not fully understood. We have examined the effects of plasma obtained from LPStreated human whole blood on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) on human endothelial cells. Stimulation of blood with 10 pg/ml of LPS is sufficient to produce plasma that induces E-selectin and ICAM-1 expression, while direct induction by LPS alone requires a 100-fold higher concentration. Characteristics for the plasma-induced adhesion molecule expression were similar to the LPS-induced production of TNF-a and IL-1b in blood. A complete inhibition of E-selectin and ICAM-1 expression was observed when antibodies against TNF-a and IL-1b were added to plasma prior to the incubation to endothelial cultures. Significant inhibition was even observed if antibodies were added to the cultures up until 3 h after LPSconditioned plasma. The plasma-induced adhesion molecule response could also be prevented with inhibitors of nuclear factor (NF)-kB, such as pyrollidine dithiocarbamate. These findings emphasize the central role of TNF-a and IL-1b in LPS-induced endothelial activation and suggest that simultaneous neutralization of these cytokines or their common pathways may, even after the initial stimulus, prevent endothelial response during sepsis.