Effects of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers

Abstract

Tumor necrosis factor α (TNF-α) plays a crucial role in the pathogenesis of the central nervous system infections. In an in vitro reconstructed blood–brain barrier model, a significant dysregulation of receptor mediated endocytosis of low density lipoproteins (LDL) and transferrin (Tf) is demonstrated at delayed phase of direct TNF-α activation. Concomitant with the increase in LDL uptake, we demonstrate a decrease of Tf-receptor mediated endocytosis. The potential role of TNF action in the differential or opposite routing of macromolecules is also characterized by a stimulation of their transcytosis. These findings may provide a new insight into the inflammatory effect of TNF-α on brain capillary endothelial cells.

Introduction

Cytokines participate in a wide variety of biological processes including growth, development and in tissue dysregulation such as inflammatory diseases, infections or trauma. In normal conditions, their expression is relatively low whereas in pathological processes a chronic production of cytokines occurs, which results in a dysregulation of local or systemic homeostasis. The large number of cells able to produce cytokines, the wide range of their biological activities and the overlapping effects of these activities, contribute to the difficulty in identifying their physiological roles.

At the brain level, the molecular mechanisms initiating and propagating the inflammatory reactions as well as the role of cytokines during these processes are not well understood. TNF-α, a proinflammatory cytokine that is produced mainly by macrophages but also by glial cells or astrocytes, appears to be involved in the pathogenesis of several infections of the central nervous system (CNS) such as bacterial meningitis (Leist et al., 1988; Waage et al., 1989), cerebral malaria (Grau et al., 1987) and human immunodeficiency virus type 1 encephalopathy (Grimaldi et al., 1991). There is also evidence that TNF-α plays an important role in neurodegenerative diseases such as multiple sclerosis (Hofman et al., 1989; Sharief and Hentges, 1991; Tsukada et al., 1991; Sharief and Thompson, 1992), Alzheimer's disease (Fillit et al., 1991) and Parkinson's disease (Mogi et al., 1994). Indeed, in such diseases TNF-α is found in serum and/or cerebrospinal fluid of patients, suggesting that this cytokine may play an important role in the development of inflammatory reactions observed during the course of these pathologies.

There is evidence that alterations of the BBB functions and cerebral endothelial cell damage occur in the development of the pathologies above-described. BBB breakdown was described in infectious diseases such as bacterial meningitis (Tuomanen et al., 1985) or in neurological disorders (Kermode et al., 1990; Petito and Cash, 1992; Mecocci et al., 1991). Cerebral capillary endothelial cells are known to be the functional and structural basis of the BBB. These endothelial cells are highly specialized cells and possess narrow tight junctions, reduced numbers of transcytotic vesicles (Reese and Karnovski, 1967), polarized enzymatic activities between the luminal and abluminal membranes (Betz et al., 1980). All these structural features of the BBB endothelium restrict the entry of hydrophilic compounds and macromolecules into the brain.

Mechanisms of the BBB permeability perturbations during inflammatory diseases and in CNS pathologies appear complex (for review, see Feuerstein et al. (1994)). Megyeri et al. (1992)have shown that TNF-α administrated into the cerebrospinal fluid (CSF) of newborn pigs, induced an extravasation of sodium fluorescein in different parts of the brain parenchyma. In the same way, Kim et al. (1992)have shown that unlike intravenous injection, intracisternal administration of TNF-α in rats is followed by a penetration of circulating albumin in CSF.

The effect of TNF-α in the blood compartment is more confusing. Using capillary depletion experiments, Gutierrez et al. (1993), found no change in the BBB permeability for albumin after intravenous administration of TNF-α. However, previous in vitro studies from our laboratory have shown that a luminal activation of differentiated brain capillary endothelial cells from coculture with astrocytes, with high concentrations of TNF-α, lead to a delayed increase in the sucrose permeability and a selective reorganization of F-actin filaments (Deli et al., 1995).

Therefore, until now the works that studied the effects of TNF-α on the blood–brain barrier focused principally on the BBB permeability changes associated with non-specific transports. However, brain capillary endothelial cells express on their surface and in a polarized manner, some specific receptors that regulate selective transport of compounds from the blood to the brain.

As lipids and iron are essential for normal growth and function of the brain cells and as the low-density lipoproteins (LDL) and the diferric transferrin (holoTf) undergo transcytosis across brain capillary endothelial cells via a receptor-mediated pathway (Dehouck et al., 1994; Dehouck et al., submitted; Descamps et al., 1996), we have chosen to study the direct effect of TNF-α on the intracellular traffic and transport of these blood-borne macromolecules at the brain capillary ECs level, all within the context of the endothelial barrier function.