Preferential expression and function of Toll-like receptor 3 in human astrocytes

https://doi.org/10.1016/j.jneuroim.2004.09.009Get rights and content

Abstract

In contrast to other tissues, the central nervous system (CNS) is essentially devoid of MHC expression and shielded from antibodies by the blood–brain barrier. Therefore, a rapid local innate immune response by resident brain cells is required to effectively fight infectious agents. This study analyzed the expression and function of Toll-like receptors (TLRs) in cultured human astrocytes. Quantitative PCR for TLRs 1 to 10 showed a basal expression of TLR3 that could be enhanced by IFN-γ, IL-1β, and IFN-β. The other TLRs were barely detectable and not inducible by the same cytokines. IFN-γ-activated astrocytes responded to TLR3 ligand poly (I:C) engagement with IL-6 production, while ligands of other TLRs, like LPS, lipoteichoic acid, peptidoglycan, flagellin, and CpG, had no effect. Poly (I:C) also triggered astrocyte production of TNF-α and the chemokines CCL2/MCP-1, CCL5/RANTES, CCL20/MIP-3α, and CXCL10/IP-10. The adapter molecules MyD88 (full length and short isoform), TIRAP/Mal, and TICAM-1/TRIF, which are required for TLR signaling, were all expressed by astrocytes. Thus, resting and activated human astrocytes express preferentially TLR3 and, upon TLR3 engagement, produce IL-6 and chemokines active on T cells, B cells, monocytes, and dendritic cells. These data indicate that astrocytes function as sentinels for viral infections in the CNS.

Introduction

Special features of the central nervous system (CNS) distinguish it from other tissues in terms of immunoreactivity. The CNS is designed to prevent any kind of accidental inflammation, which might be deleterious for the whole organism. Mechanisms to achieve this are the almost complete absence of MHC expression, a blood–brain barrier excluding antibodies, production of transforming growth factor-ß, and inhibition of microglia activity via neuronal CD200 (reviewed in (Griffin, 2003)). On the other hand, resident brain cells must be able to rapidly alert the immune system to fight infectious agents. The limited access of components of the adaptive immune system to the normal CNS suggests that an effective intracerebral innate immune response is needed to start locally the fight against infectious agents.

The innate immune system has the extraordinary feature to sense a broad spectrum of pathogens using invariant receptors. These receptors are specific for elements called pathogen-associated molecular patterns shared by large groups of microorganisms (Medzhitov and Janeway, 2002). Upon recognition, immune cells get activated, mount a strong response against infected cells, and subsequently activate the adaptive immune system (Takeda et al., 2003, Farina et al., 2004). Toll-like receptors (TLRs) mediate microbial recognition in several organisms including humans, mice, and fly. For some of them, the TLRs ligands were identified. TLR2 recognizes bacterial lipoproteins, peptidoglycan (PGN), and lipoteichoic acid (LTA) from Gram-positive bacteria, and zymosam from fungi, and can associate with TLR1 and TLR6 for functional responses to mycobacterial lipopeptides. TLR3 recognizes viral double-stranded (ds) RNA and synthetic dsRNAs, such as polyinosinic-polycytidylic acid [Poly (I:C)]. TLR4 binds LPS from Gram-negative bacteria, while TLR5 recognizes flagellin, the primary protein component of bacterial flagella. TLR7 and TLR8 have recently been identified to recognize viral single stranded RNA (Diebold et al., 2004, Heil et al., 2004, Lund et al., 2004). TLR9 recognizes CpG motifs within bacterial DNA (Takeda et al., 2003) and the recently discovered TLR11 recognizes uropathogenic bacteria (Zhang et al., 2004).

The expression of TLRs has been mainly analyzed in immune cells, which differ with respect to TLR expression in a cell-type and differentiation-dependent manner (Muzio et al., 2000, Hornung et al., 2002). TLRs have also been found in parenchymal cells. For example, TLR5 is expressed at the basolateral surface of intestinal epithelial cells (Gewirtz et al., 2001). In renal epithelial cells, TLR2 and TLR4 expression is induced by IFN-γ and contributes to the detection of bacterial invasion in the lumen of tubules (Wolfs et al., 2002). TLR4 has been detected in cultured dermal endothelial cells (Faure et al., 2000) and resident corneal epithelial cells, and reported to play an important role in the inflammatory process in river blindness (Saint et al., 2002). Expression of various TLRs was observed on retinal pigment epithelial cells (Kumar et al., 2004).

Cultured mouse and human microglial cells were recently shown to express various TLRs (Bsibsi et al., 2002, Kielian et al., 2002). Human astrocytes were reported to express TLR-2 and TLR3 (Bsibsi et al., 2002), while in mouse astrocytes TLR2, TLR4, TLR5, and TLR9 were investigated and found expressed (Bowman et al., 2002; Esen et al., 2004). There is increasing interest in innate immune responses in the CNS, since TLR-mediated effects on glial cells have been linked not only to fighting bacterial infections (Esen et al., 2004), but also to neurotoxicity (Lehnardt et al., 2003, Iliev et al., 2003), neuroprotection (Nguyen et al., 2002), oligodendrocyte injury (Lehnardt et al., 2002), and CNS immunopathology (Dalpke et al., 2002).

Astrocytes are the most abundant cell type in the central nervous system. They provide support to neurons, modulate synaptic activity, participate in the formation of the blood–brain barrier, and interact with immune cells in multiple ways (Weber et al., 1994, Meinl et al., 1994, Aloisi et al., 2000, Dong and Benveniste, 2001).

The aim of this study was to quantify astrocytic expression of TLRs 1–10, learn how their expression is regulated, identify TLR-adapter molecules expressed by astrocytes, and describe functional responses of human astrocytes to TLR engagement.

Section snippets

Reagents

The following reagents were used: human IFN-γ (500 U/ml, Roche, Mannheim, Germany), IL-1β (100 ng/ml, R&D, Wiesbaden-Nordenstadt, Germany), IFN-β (1000 U/ml, Betaferon, Schering, Berlin, Germany), Flagellin from Helicobacter pylori (5 μg/ml, IBT, Reutlingen, Germany), Peptidoglycan from Staphylococcus aureus (PGN, 5 μg/ml, Fluka, Sigma-Aldrich, Schnelldorf, Germany), Lipoteichoic acid from S. aureus (LTA, 5 μg/ml), LPS from E-Coli 0111:B4 (300 ng/ml, Sigma-Aldrich), CpG ODN (5 μM, 5′-atcgactctcg

Astrocytes express preferentially TLR3

Cultured human astrocytes were analyzed for expression of TLRs 1–10 by quantitative PCR. Table 1 shows a representative experiment. TLR3 was the only TLR with a consistent basal expression. TLR3 mRNA levels were robustly increased after stimulation of astrocytes with IFN-γ and IFN-β, and to a lesser extent with IL-1β. None of these cytokines induced the expression of the other TLRs (Table 1). Experiments were performed with two different human astrocyte cultures with similar results (Table 1,

Discussion

This study analyzes the role of astrocytes in innate immune responses within the CNS. Astrocytes are the most numerous glial cell type and have a fundamental role in maintaining CNS homeostasis. In addition, astrocytes actively participate in the regulation of inflammation and immunity by producing cytokines and chemokines, while their role as antigen presenting cells remains controversial (Weber et al., 1994, Aloisi et al., 2000, Dong and Benveniste, 2001).

Real-time PCR for TLRs 1–10 revealed

Acknowledgements

We thank Mrs. D. Zech for technical assistance, and Prof. R. Voltz and Dr. A. Flügel for reading the manuscript. We are grateful to Prof. R. Hohlfeld for continuous support. This work was supported by the DFG (SFB 571 and GRK688). The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.

References (60)

  • S. Lee et al.

    CpG oligodeoxynucleotides induce expression of proinflammatory cytokines and chemokines in astrocytes: the role of c-Jun N-terminal kinase in CpG ODN-mediated NF-kappaB activation

    J. Neuroimmunol.

    (2004)
  • M. Matsumoto et al.

    Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling

    Biochem. Biophys. Res. Commun.

    (2002)
  • P.J. Nelson et al.

    Chemokines, chemokine receptors, and allograft rejection

    Immunity

    (2001)
  • C. Pasare et al.

    Toll-like receptors: balancing host resistance with immune tolerance

    Curr. Opin. Immunol.

    (2003)
  • A. Rousselet et al.

    Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striatal astroglia

    Dev. Biol.

    (1988)
  • D. Theil et al.

    Latent herpesvirus infection in human trigeminal ganglia causes chronic immune response

    Am. J. Pathol.

    (2003)
  • G.A. Viglianti et al.

    Activation of autoreactive B cells by CpG dsDNA

    Immunity

    (2003)
  • L. Alexopoulou et al.

    Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3

    Nature

    (2001)
  • F. Aloisi et al.

    Astrocyte cultures from human embryonic brain: characterization and modulation of surface molecules by inflammatory cytokines

    J. Neurosci. Res.

    (1992)
  • E. Ambrosini et al.

    Astrocytes are the major intracerebral source of macrophage inflammatory protein-3alpha/CCL20 in relapsing experimental autoimmune encephalomyelitis and in vitro

    Glia

    (2003)
  • C.C. Bowman et al.

    Cultured astrocytes express Toll-like receptors for bacterial products

    Glia

    (2003)
  • M. Bsibsi et al.

    Broad expression of Toll-like receptors in the human central nervous system

    J. Neuropathol. Exp. Neurol.

    (2002)
  • E. Cario et al.

    Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease

    Infect. Immun.

    (2000)
  • A.H. Dalpke et al.

    Immunostimulatory CpG-DNA activates murine microglia

    J. Immunol.

    (2002)
  • S.S. Diebold et al.

    Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA

    Science

    (2004)
  • Y. Dong et al.

    Immune function of astrocytes

    Glia

    (2001)
  • A.D. Edwards et al.

    Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+DC correlates with unresponsiveness to imidazoquinolines

    Eur. J. Immunol.

    (2003)
  • U. Eriksson et al.

    Dendritic cell-induced autoimmune heart failure requires cooperation between adaptive and innate immunity

    Nat. Med.

    (2003)
  • N. Esen et al.

    Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

    J. Neurochem.

    (2004)
  • C. Farina et al.

    Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines

    Int. Immunol.

    (2004)
  • Cited by (228)

    • Immunotherapeutic interventions in Parkinson's disease: Focus on α-Synuclein

      2022, Advances in Protein Chemistry and Structural Biology
    View all citing articles on Scopus
    View full text