Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels.
We have compared the induced expression of E-selectin in primary cultures of rat brain microvascular endothelial cells (EC), pericytes and in non-CNS microvascular endothelium stimulated with the cytokines, IL-1beta (20 ng/ml), and tumor necrosis factor (TNF)-alpha (75 ng/ml). Expression was studied at both the protein and mRNA levels. Fluorescence in-situ hybridization (FISH) was used to examine de novo synthesis of E-selectin mRNA. Laser cytometric analysis was used as a novel approach to the quantitaion of FISH. In-situ hybridization was performed using two PCR-generated probes. The first probe (517 bp) spanned the lectin and epidermal growth factor (EGF)-like domain. The second probe (562 bp) spanned the CR3, 4, and 6 domains. E-selectin-specific mRNA was localized to the perinuclear regions of the EC. Both cytokines, IL-1beta and TNF-alpha significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1beta induced higher E-selectin mRNA levels than TNF-alpha. The maximum number of mRNA-positive cells was observed after stimulation for 4--6 h. Surface protein expression was sustained for up to 48 h following addition of cytokine. This was in contrast to the transient expression in non-CNS EC indicating that pure primary CNS EC display slightly different kinetics of E-selectin expression than non-CNS EC.
Wagnerova
Jarmila
J
Wayne State University School of Medicine, Department of Neurology, Division of Neuroimmunology, Detroit Medical Center, 421 E. Canfield Ave, Detroit, MI 48201, USA.
Cervenakova
Larisa
L
Balabanov
Roumen
R
Zitron
Ian
I
Dore-Duffy
Paula
P
D000818:Animals; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000235:genetics; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000502:physiology*; D008297:Male; D008833:Microcirculation; D020286:Pericytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D018076:DNA, Complementary; D019040:E-Selectin; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha