SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics

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Abstract

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Introduction

The blood-brain (BBB) and blood-retinal (BRB) barriers form a selective cellular interface between the blood and the central nervous system (CNS). These barriers are important in controlling the passage of molecules and cells to and from the neural parenchyma and thus in maintaining homeostasis. In the brain, the endothelial cells of the cerebral vasculature that constitute the BBB, form tight intercellular junctions which, in conjunction with minimal pinocytosis and a lack of pores, leads to impermeable vessels with very high electrical resistance's (Crone and Olesen, 1982; Butt and Jones, 1992). In the retina, the BRB is comprised of two distinct cell types that are separated anatomically. The retinal vascular endothelia, that feed the anterior portion of the retina, are thought to be identical to those of the brain (Greenwood, 1992a; Towler et al., 1994). The retinal pigment epithelium (RPE), however, overly the permeable vessels of the choroidal circulation and form the posterior barrier by virtue of their tight apical junctions and in this respect are similar to the tight epithelial cells of the choroid plexus. Together these two cell types constitute the BRB.

It is clear that the specialised properties of CNS endothelia is not restricted to the expression of tight junctions and the formation of a physical barrier. Other properties of the endothelia also contribute to the specialised nature of the barrier, in particular the unique distribution and expression of many surface molecules that are absent on endothelia from other organs. This can be exemplified by the expression of molecules such as the high affinity glucose transporter (GLUT-1), the transferrin receptor and the product of the multidrug resistance gene, P-glyco-protein (Pgp) as well as the absence of other antigens which are expressed on non-CNS endothelia such as OX-43 (in the rat) and PAL-E (in humans).

Endothelia from the brain and retina also differ from peripheral endothelia in their ability to capture circulating leucocytes, being considerably less adhesive than non-CNS vascular beds (Hughes et al., 1988; Male et al., 1990; Wang et al., 1993). During immune-mediated diseases of the CNS, however, the vascular endothelia play a critical role in recruiting leucocytes from the circulation (Male et al. (1990), Male et al. (1994); Greenwood and Calder, 1993; Greenwood et al., 1995; Calder and Greenwood, 1995; Devine et al., 1996a) and in influencing lymphocyte function (McCarron et al., 1985; Risau et al., 1990; Wang et al., 1995; Bourdoulous et al., 1995).

Both the normal and pathological functions of cells of the BBB and BRB have been the subject of a substantial amount of research. Until recently, most investigations were restricted to in vivo studies but, with the experimental flexibility offered by in vitro methods, techniques have been developed to isolate and culture CNS-derived endothelial cells (for reviews see Greenwood, 1991; Joo, 1996). These techniques, however, have proved to be both difficult and time consuming as CNS-derived endothelia and RPE are phenotypically unstable in long-term culture, remain impure and have low plating efficiency. As a result of this phenotypic instability, most investigations are confined to the use of primary cultures. Techniques are now available, however, to immortalise cells and the development of such cells from the BBB and BRB will be of considerable use in investigating the properties of these specialised cellular barriers. However, this approach will be of value only if the immortalised cells retain the characteristics of primary cultures over many passages or, if panels of different cell lines can be generated which together encompass the characteristics of primary cultures.

In a series of recent reports an immortalised rat brain endothelial cell line (RBE4) has been described that has proved to be a valuable resource for studying some of the properties of cerebral endothelia (Roux et al., 1994; Bourdoulous et al., 1995; Abbott et al., 1995). These cerebral endothelia, which were immortalised by transfection with pE1A-neo which encodes the adenovirus 2 E1A gene, retain many of their in vivo characteristics and are proving to be useful in a number of investigations. Similarly, there has also been a recent report of an SV40 large T immortalised RPE cell line (RPE-J) which retain many of the characteristics of RPE primary cultures (Nabi et al., 1993). However, since the techniques used to immortalise cells are based on random chromosomal integration and copy number of the immortalising gene, the resulting lines are likely to differentially reflect the characteristics of endothelial or RPE primary cultures. It is therefore important that further cell lines are produced so that those retaining particular characteristics can be identified.

Here we describe the development and characterisation of immortalised cells derived from primary cultures of both rat blood-brain and blood-retinal barriers using a temperature sensitive form of the SV40 large T antigen which has previously been used to immortalise rodent cells (Jat et al., 1986). The production of stable cell lines that retain many of the characteristics of primary cultures will be of great use to those investigating the properties of the blood-tissue barriers of the CNS including their important role in the pathogenesis of immune-mediated CNS disease.

Section snippets

Materials

Rat recombinant IFN-γ was obtained from Holland biotechnology BV (Leiden) and mouse TNFα was obtained from Genzyme (Kent). Mouse anti-rat intercellular adhesion molecule-1 (ICAM-1; 1A29) and mouse anti-rat platelet endothelial cell adhesion molecule-1 (PECAM-1; 3A12) monoclonal antibodies (mAbs) were obtained from Serotec (Oxford). The anti-rat vascular cell adhesion molecule-1 (VCAM-1; 5F10) was a generous gift from Dr. R. Lobb (Biogen, MA, USA). The anti-rat MHC class II I-A (OX-6) and MHC

SV40 large T transfection of rat cerebral and retinal endothelia and RPE

Transfected cells selected by sustained growth in G418 produced parent lines of brain and retinal endothelia which were designated the codes GP8 and JG2, respectively. From these parent lines a number of clones were produced. Using morphological criteria one clone from each parent line was selected for expansion and investigation. The cerebral endothelial clone was designated as GP8.3 and the retinal endothelial clone as JG2.1. The parent cell line of the transfected RPE cells was designated

Discussion

The infection of primary cultures of both rat brain and retinal endothelial cells and rat RPE cells with a recombinant retrovirus encoding the SV40 large T-antigen and a selectable neomycin resistance marker has generated G418-resistant parent cell lines and cell clones which express the large T antigen. In contrast to human cells where transfection of the SV40 large T antigen has been reported to only result in an extended life span of umbilical vein endothelial cell cultures (Fickling et al.,

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