Mononuclear Cells Induce E-selectin Gene Expression in Porcine Endothelial Cells

Cha, Young Joo; Chae, Seok Lae; Kim, Cheol Ho

Abstract

BACKGROUND
E-selectin, a cell adhesion molecule for most leukocytes, is known to be expressed exclusively on the cytokine-stimulated endothelial cells (ECs). E-selectin is involved in the adhesion of neutrophils, monocytes and a subset of T cells to inflammatory, but not unstimulated endothelium. E-selectin expression has been demonstrated in transmedial vessels in atherosclerotic plaques, where the intensity of staining correlated with that of infiltrated mononuclear cells (MNCs). In this study we have investigated whether MNCs interacting with ECs in co-culture system could induce the transcriptional activation of the E-selectin gene, and influence the response to cytokine stimulation.
METHODS
Porcine ECs derived from aorta in fourth to fifth passage were grown in medium 199 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum and penicillin-gentamicin (100U/mL and 20 microgram/mL, respectively). MNCs were isolated using density gradient centrifugation. Subconfluent ECs in 60-mm plate were overlaid with MNCs suspension (1x106) and co-cultured. In some experiments, interleukin-1beta (IL-1beta) was added to the culture plate at concentration of 10U/mL. Total RNA from ECs co-cultred with MNCs was isolated, reverse transcribed forming cDNA and amplified using primers. The PCR reaction product was run on a 1% agarose gel and the gels photographed under UV light.
RESULTS
Co-culture of MNCs on ECs led to significant induction of E-selectin expression. E-selectin mRNA expression on ECs was induced after 3hrs and sustained at 24hrs following co-culture with MNCs, comparing the results that E-selectin mRNA expression on ECs was induced after 12 hrs and disappeared at 24hrs when ECs was stimulated only by IL-1beta. On the other hands, strong E-selectin mRNA expression on ECs was induced after 3hrs and sustained after 24 hrs following co-culture with MNCs and stimulated by IL-1beta.
CONCLUSIONS
MNCs can interact with ECs to produce sustained up-regulation of E-selectin, as well as enhancing the response to IL-1beta.