title: Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers.
abstract: The deposition of circulating immune reactants in blood vessels, an important event in the pathogenesis of certain types of vasculitis, requires an increase in permeability in the endothelial monolayer. An in vitro model to examine the integrity of endothelial cell monolayers and their response to inflammatory mediators has been developed. Human umbilical vein endothelial cells were grown to confluence on an FITC-labelled matrix and monolayer integrity was assessed by the exclusion of a 125I-anti-FITC antibody. Alteration in endothelial monolayer permeability was associated with an increase in uptake of 125I-anti-FITC antibody, expressed as a percentage of the maximal uptake of antibody on to FITC-matrix from which endothelial cells had been stripped. We determined the effects on endothelial monolayer permeability of acute agonists (thrombin and histamine), cytokines (tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 and IL-4) and combinations of acute agonists and cytokines. Addition of thrombin in concentrations ranging from 0.5 to 15 U/ml led to an increased uptake of 125I-anti-FITC antibody from 2% to 15% relative to unstimulated endothelium. For other agonists and cytokines the increases in permeability were: (i) histamine (50-400 pmol/ml) increased uptake 5-22%; (ii) TNF (12.5-100 ng/ml) increased uptake 2-12%; (iii) IFN-gamma (125-250 U/ml) increased uptake 1.5-3%. IL-1 beta (50-100 U/ml) and IL-4 (50-100 U/ml) had no effect. Synergistic interactions on endothelial monolayer permeability were seen with the following combinations: (i) IL-4 (100 U/ml) and TNF (12.5 ng/ml) uptake 11%; (ii) IL-4 (100 U/ml) and IFN-gamma (125 U/ml) uptake 6.5%; (iii) TNF (12.5 ng/ml) and IFN-gamma (125 ng/ml) uptake 7%; (iv) thrombin (0.5 U/ml) and histamine (50 pmol/ml) uptake 13.5%; and (v) TNF (12.5 ng/ml) and thrombin (0.5 U/ml) uptake 8.5%. These observations suggest that interactions between cytokines and acute inflammatory mediators such as thrombin and histamine may be important in determining whether immune complexes are deposited in vessel walls. This model system may now be useful for the further investigation in vitro of the mechanisms involved in the pathogenesis of immune complex-mediated vascular damage.
authors.affiliation: Rheumatology Unit, RPMS, Hammersmith Hospital, London, UK.
authors.forename: H L
authors.initials: HL
authors.lastname: Beynon
authors.forename: D O
authors.initials: DO
authors.lastname: Haskard
authors.forename: K A
authors.initials: KA
authors.lastname: Davies
authors.forename: R
authors.initials: R
authors.lastname: Haroutunian
authors.forename: M J
authors.initials: MJ
authors.lastname: Walport
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004357:Drug Synergism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides; D051379:Mice; D013917:Thrombin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D006632:Histamine; D007371:Interferon-gamma; D013917:Thrombin