Purchase this article with an account.
K.G. Shadrach, C.A. de la Motte, J. Drazba, G. Hoppe, P.D. Senanayake, J.G. Hollyfield; Monocyte Adhesion to Retinal Pigment Epithelium Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2876.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To evaluate the binding of monocytes by Hyaluronan (HA) on retinal pigment epithelium (RPE) cells under hyperglycemic stress. HA is synthesized by the RPE. Elevated glucose concentration leads to activation of HA synthase HAS2 and stimulation of HA synthesis. Recent studies have suggested that HA may play a role in the inflammatory response component of diabetic complications.
Confluent ARP19 cells were cultured on cover slips for 6 days in DMEM: F12, 15 mM HEPES medium containing 10% fetal bovine serum, penicillin–streptomycin–glutamine. Three glucose concentrations were tested: 1,5 or 10 mg/ml. The medium was changed every two days. On day 6, U937 monocytic binding to unstimulated or the viral mimic [(polyinosinic acid:polycytidylic acid (polyI:C)] (20 µ g/ml) or tumor necrosis factor alpha (TNF–α ) (10ng/ml) by RPE cells was evaluated. RPE cells in the respective medium were treated with PolyI: C or TNF–α 18 hours at 370 C before the assay. The incubation medium was aspirated from the RPE cells, and 106 leukocytes were added to each well. The binding phase of the assay was done at 40 C for 1 hour. Subsequently, the RPE cells on cover slips contained in wells were washed with medium and rinsed with Hank’s balanced salt solution. The RPE cells were then fixed in –200C methanol, air–dried and observed by phase contrast microscopy (X100).
TNF–α treatment resulted in monocyte adhesion to RPE cells cultured in 10–mg/ml glucose. U937 cells appeared as bright spheres on top of the RPE cells. Whereas 1 mg/ml, 5 mg/ml and 10 mg/ml glucose did not stimulate adhesion of monocytes to RPE cells. PolyI: C treatment did not provoke the binding of monocyte to RPE cells.
High glucose and TNF–α stimulation were both required for HA mediated monocyte adhesion to RPE cells.
This PDF is available to Subscribers Only