Expression and regulation of toll-like receptors in cerebral endothelial cells

Abstract

Cerebral endothelial cells – the principal components of the blood–brain barrier (BBB) – fulfill several important functions in the central nervous system (CNS). They form an active interface between blood and neuronal tissue and play a key role in the maintenance of the homeostasis of the CNS. Infections caused by different pathogens are often associated with systemic symptoms and may compromise the functional integrity of the BBB as well. In the mediation of the systemic effect of pathogens Toll-like receptors (TLRs) play a significant role. TLRs are a type of pattern recognition receptor and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules. TLRs are broadly distributed on cells of the immune system and function as primary sensors of invading pathogens. There is also growing experimental evidence indicating that Toll-like receptors are expressed on different non-immune cell types as well, like epithelial or endothelial cells. Here we demonstrate the expression of TLR2, TLR3, TLR4 and TLR6 on rat and human cerebral endothelial cells. Oxidative stress significantly upregulated the expression of these receptors whereas TNF-alpha upregulated the expression of TLR2 and TLR3. Furthermore we have shown, that activation of TLR2/6 leads to an increased permeability which is accompanied by a downregulation of occludin and claudin-5 expression and disappearance of these tight junction proteins from the cell membrane. Changes in occludin expression and localization could be inhibited by the ERK1/2 inhibitor U0126. Our results suggest a significant role of the cerebral endothelium in mediation of the neural effects of different inflammatory processes.

Introduction

By forming a single-cell layer lining the blood vessels of the brain, cerebral endothelial cells (CECs) constitute the principal components of the blood–brain barrier (BBB). They form an active interface between blood and neuronal tissue and play a key role in the maintenance of the homeostasis of the central nervous system (CNS). It is well established that under pathological conditions (e.g., inflammatory disorders, cerebral ischemia and subsequent reperfusion, brain tumors, trauma, diabetes) an increase in BBB permeability may occur, which may lead to a disturbed homeostasis of the CNS with severe consequences. Among the large number of functions fulfilled by CECs perhaps the most important is the barrier function.

The permeability of the endothelial barrier is largely determined by the integrity of the tight junctions and adherens junctions between endothelial cells. In this respect the transmembrane proteins of the junctional complexes including occludin, members of the claudin-family and the junctional adhesion molecules (JAMs) may have a special importance. An important role in the organization of the structure of tight junctions play the zonula occludens (ZO) family of scaffolding proteins: ZO-1, ZO-2 and in epithelial cells ZO-3 as well. In addition adherens junctions, especially their transmembrane proteins, the cadherins are also able to participate in the regulation of BBB permeability (Pal et al., 1997). Furthermore, a complex system of signaling molecules is expressed in CECs making possible the precise regulation of endothelial functions (Deli et al., 1993, Fábián et al., 1998, Krizbai and Deli, 2003).

Since cerebral endothelial cells are in direct contact with blood they are in the forefront of the defense system of the CNS and are of crucial importance in sensing and responding to stress factors. Infections caused by different pathogens are often associated with multiorgan symptoms involving the CNS as well. An important factor in the appearance of CNS symptoms may be the compromised functional integrity of the BBB as well. In the mediation of the systemic effects of pathogens Toll-like receptors (TLRs) play a significant role.

Toll-like receptors are important recognition receptors of the innate immune system: they are evolutionarily conserved in both the invertebrate and vertebrate lineages. To date, 13 members of the TLR family have been identified in mammals. Mice express all TLRs (TLR1–TLR13), but humans express only 10. However, equivalent forms of certain human TLRs are not present in all other mammalian species. These differences between species may complicate the modelling of human innate immunity by experimental animals. Toll-like receptors function as pattern recognition receptors (PRR). They recognize molecular patterns that are well conserved among pathogens but distinguishable from host molecules. These so called pathogen associated molecular patterns (PAMPs) include bacterial lipopolysaccharides (LPS), lipoproteins, lipopetides, zymosan from fungi and various viral DNAs and RNAs (for review see: Akira and Hemmi, 2003). Beside PAMPs certain TLRs also sense products of damaged tissue (damage associated molecular patterns, DAMPs), such as heat-shock proteins, fragments of hyaluronan and fibronectin (Vabulas et al., 2001, Okamura et al., 2001, Scheibner et al., 2006).

Both PAMPs and DAMPs are able to induce inflammation therefore TLRs may be important players in the initiation of the inflammatory response in both cases. There is a growing body of evidence indicating that TLRs can also be activated by proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (Faure et al., 2001).

The engagement of TLRs by different ligands triggers the activation of signaling cascades, leading to the induction of genes involved in antimicrobial host defense. After ligand binding TLRs dimerize and undergo conformational changes required for the recruitment of Toll/interleukin-1 receptor (TIR) domain-containing adaptor molecules to the TIR domain of the TLR. The differential recruitment of these adapter proteins by different TLRs form the basis for the specificity in the signaling process activated by them. This recruitment of adaptors triggers the cascade of signaling pathways and ultimately the activation of transcription factors such as nuclear-factor kappa B (NF-κB) and members of the interferon-regulatory factor (IRF) family. Furthermore, this recruitment also activates mitogen-activated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) 1/2 and c-jun NH₂-terminal kinase (JNK) which activate the AP-1 (activating protein 1) transcription factors. These transcription factors induce the transcription of inflammatory cytokines, type I interferons and chemokines (for review see: Kawai and Akira, 2006).

TLRs are broadly distributed on cells of the immune system and function as primary sensors of invading pathogens. There is also growing experimental evidence that Toll-like receptors are expressed on different non-immune cell types as well, like epithelial or endothelial cells (Koff et al., 2008, Loos et al., 2006, Pryshchep et al., 2008). However, few data is available about the expression and function of TLRs in brain endothelial cells. Several recent studies including ours suggested that at least TLR2 (Ziegler et al., 2007) TLR3 (Fischer et al., 2009) and TLR4 (Veszelka et al., 2007, Singh et al., 2007) may be functionally active in the brain endothelium and could be involved in mediation of pathological processes.

Despite the crucial role played by brain endothelial cells in protecting the CNS little is known about the innate mechanisms of endothelial cell activation by pathogens and the receptors that mediate and regulate these responses. The aim of the present study is to identify the TLRs expressed on cerebral endothelial cells and to evaluate the effect of their activation by different factors that occur at the level of the BBB.