Requirement of Cell-Cell Contact for up-Regulation of IL-8 by Neutrophils in Endothelial Cell Cultures

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Previously we have shown that the IL-8 production in cultured endothelial cells was up-regulated by neutrophils. Here we further study the neutrophil-endothelial cell interaction focusing on a cell-cell contact requirement. Human neutrophils were isolated by gradient centrifugation using 1-Step Polymorphs (Accurate Chemical) and either untreated or incubated in TNF-α-coated wells (500U/well) for 3 hr prior to culture or membrane isolation. Human coronary artery endothelial cells (HCAEC) and EGM medium for their growth were purchased from Clonetics. The co-cultures were carried out by adding 2 ml of neutrophils (2 x 106 cells/ml) or membrane to the wells containing confluent HCAEC cultured in a 12-well plate. The supernatants were harvested after a 17 hr incubation for IL-8 assays by Elisa. The membrane from TNF-α-treated neutrophils at 200 (p = .002), 100 (p < .005) and 50 (p = .01) ug/ml significantly up-regulated the IL-8. production in a dose response fashion, while that of the untreated neutrophils did not exhibit any effect. Separation of HCAEC and neutrophils by a membrane (0.22 μm) prevented the IL-8 induction. The results suggest that up-regulation of IL-8 production by neutrophils requires cell-cell contact.

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