Korean J Hematol 2003; 38(3):
Published online September 30, 2003
© The Korean Society of Hematology
혈관내피세포에서 단핵백혈구에 의한 세포부착분자 E-selectin의 발현
차영주, 채석래, 김철호
중앙대학교 의과대학 진단검사의학교실,
서울대학교 의과대학 내과학교실
Mononuclear Cells Induce E-Selectin Gene Expression in Porcine Endothelial Cells
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 neurotrophils, 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 sysem 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㎍/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-1β (IL-1β) 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 an 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-1β. On the other hands, strong E-seletin mRNA expression on ECs was induced after 3hrs and sustained after 24 hrs following co-culture with MNCs and stimulated by IL-1β.
Conclusions : MNCs can interact with ECs to produce sustained up-regulation of E-selectin, as well as enhancing the response to IL-1β.
Keywords Endothelial cells; E-selectin; Mononuclear cells; IL-1β
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Korean J Hematol 2003; 38(3): 183-187
Published online September 30, 2003
Copyright © The Korean Society of Hematology.
혈관내피세포에서 단핵백혈구에 의한 세포부착분자 E-selectin의 발현
차영주, 채석래, 김철호
중앙대학교 의과대학 진단검사의학교실,
서울대학교 의과대학 내과학교실
Mononuclear Cells Induce E-Selectin Gene Expression in Porcine Endothelial Cells
Young Joo Cha, Seok Lac Chae, Cheol Ho Kim
Department of Laboratory Medicine, Chung Ang University, College of Medicine, Seoul, Korea
Department of Internal Medicine, Seoul National University, College of Medicine, Seoul, Korea
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 neurotrophils, 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 sysem 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㎍/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-1β (IL-1β) 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 an 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-1β. On the other hands, strong E-seletin mRNA expression on ECs was induced after 3hrs and sustained after 24 hrs following co-culture with MNCs and stimulated by IL-1β.
Conclusions : MNCs can interact with ECs to produce sustained up-regulation of E-selectin, as well as enhancing the response to IL-1β.
Keywords: Endothelial cells, E-selectin, Mononuclear cells, IL-1β
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