Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels

Abstract

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-1β (20 ng/ml), and tumor necrosis factor (TNF)-α (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-1β and TNF-α significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1β induced higher E-selectin mRNA levels than TNF-α. 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.

Introduction

E-selectin (CD 62E) is a member of the selectin family of cell adhesion glycoproteins which are involved in facilitating leukocyte adhesion and subsequent transmigration across the endothelium to the tissue [1]. E-selectin is expressed exclusively on endothelial cells (EC) in response to inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) [1], [2], and is by definition an EC activation marker. E-selectin contributes to the adhesion cascade in the early stages of inflammation by mediating the rolling of leukocytes on the surface of activated EC, slowing them down and allowing them to adhere more firmly. E-selectin mediates the recruitment and adhesion of polymorphonuclear leukocytes, monocytes, a subpopulation of T lymphocytes [3], [4], [5], basophils, and eosinophils [6]. These cells bind E-selectin through cell surface carbohydrate structures, mainly sialyl Lewis X and related oligosaccharides [7].

The expression of E-selectin in human umbilical vein endothelial cells (HUVEC) in vitro is transient and regulated transcriptionally. Peak expression occurs within 4–6 h after activation, reaching basal levels after 16–24 h [1]. The expression of E-selectin in vivo [8], [9], [10], [11], in monocyte/EC co-culture systems [12], in the presence of inhibitors of HMG-1(Y) DNA binding [13], and in the presence of CNS microvascular pericytes [10] can be sustained. Human and rat E-selectin have been cloned and the gene sequence characterized [5], [14]. The molecule is composed of an N-terminal lectin domain followed by an epidermal growth factor (EGF)-like domain, six complement regulatory (CR1-6)-like repeats, a transmembrane region, and a short cytoplasmic sequence. Rat E-selectin molecules [14] show partial structural homology with human E-selectin, but lacks the CR5 domain.

Much of what is known of the E-selectin molecule and its role in microvascular function comes from studies using cultured HUVEC lines. These cells are large vessel EC, highly dedifferentiated, and have lost tissue and organ specificity. Recent studies have shown that cytokine-induced expression of adhesion molecules in EC is organ-specific and that ECs differentially express adhesion molecules depending on the size of the blood vessels [15]. It is intriguing to speculate that specialized endothelial differentiation as seen in CNS EC may have unique influences on the expression of E-selectin genes in a given tissue. The expression of E-selectin in the rat CNS has not been thoroughly investigated despite the extensive use of the rat in the study of inflammation and CNS inflammatory diseases [16], [17], [18]. As a first step in the investigation of organ- and vessel-specific E-selectin gene regulation, we have investigated cytokine-induced expression of E-selectin in pure primary rat CNS/primary microvessel EC and in primary microvascular pericytes. EC and PC cultures were used at a time when cells still retained characteristics of their blood brain barrier (BBB) origin (within 4 days of culture), and were devoid of contaminating cell populations. Expression was studied at the level of de novo mRNA synthesis by means of fluorescence in-situ hybridization (FISH) using PCR-generated probes. The first probe spanning the regions of lectin and EGF-like domains, the second the CR3, 4 and 6 domains. Results indicate that quantitation of FISH using laser cytometry is feasible and accurate. Further, results show that expression of E-selectin within the CNS endothelium is cell-specific and that CNS EC differentially respond to cytokine when compared to non-CNS EC.