Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis

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Abstract

It is well known that microglial cells perform a key role in mediating inflammatory processes, which are associated with neurodegenerative diseases such as multiple sclerosis (MS). In this study, we report that CD40 expression on microglia is greatly enhanced by a low dose (10 U/ml) of IFN-γ. We also find that ligation of microglial CD40 by CD40L triggers a significant production of TNF-α. Activation of microglia by ligation of CD40 in the presence of IFN-γ results in cultured cortical neuronal injury, which is markedly attenuated by blockade of the CD40 pathway or neutralization of TNF-α. Finally, we find significant levels of IFN-γ and TNF-α in the medium of co-cultured activated CD4+ T cells and microglial cells, showing that microglia can supply the CD40 receptor to activated CD4+ T cells and suggesting that this cellular interaction is a key event in MS pathophysiology.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelinating white matter lesions (Martin et al., 1992). Co-localization of reactive CD4+ T cells and activated microglia has been found in active MS lesions (Schluesener et al., 1996; Gay et al., 1997), suggesting that these cell types might interact in bringing about the MS process; however, the molecular mechanisms leading to microglial activation in MS remain unknown. The pro-inflammatory cytokine IFN-γ, secreted by activated T cells, has been shown to be released in large amounts in MS patients (Beck et al., 1988; Grimaldi and Martino, 1995; Sarchielli et al., 1997). However, little is known about the immune mechanisms leading to elevated IFN-γ levels, although a recent study suggested that IL-12 might lead to IFN-γ secretion in MS (Balashov et al., 1997).

Interaction of CD40, an important cellular signaling and activation antigen, with CD40L, which is found on activated CD4+ T lymphocytes, plays a central role in both humoral and cellular immunity (Armitage et al., 1992; Lane et al., 1992). For example, CD40L deficient mice fail to generate antigen-specific T cell responses and blockade of the CD40–CD40L interaction in vivo inhibits T cell-dependent IL-12 secretion by antigen-presenting cells (Foy et al., 1994; Xu et al., 1994; Grewal et al., 1996; Stuber et al., 1996; Carayanniotis et al., 1997). High levels of CD40L-expressing cells have been found to be co-localized with CD40-bearing cells in active MS lesions (Gerritse et al., 1996), suggesting that the CD40–CD40L interaction might be a mediator for immune-induced MS lesions. Further, inhibition of the CD40–CD40L interaction in an animal model of MS retards the development of the disease (Gerritse et al., 1996). However, the temporal sequence of molecular events surrounding activation of microglia and the possible mechanism for interaction between microglia and CD4+ T cells remains unknown. In order to dissect out the cellular mechanisms leading to microglial activation and increased levels of IFN-γ observed in MS, we examined the potential role that the CD40–CD40L interaction might play in these processes.

Section snippets

Microglial cell culture and reagents

Murine microglial cell lines (N9, N60) were grown in RPMI medium supplemented with 5% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 0.1 μg/ml streptomycin and 0.05 mM 2-mercaptoethanol according to previously described methods (Corradin et al., 1993). The N9 cell line was previously characterized by Corradin et al. (1993), and found to express the same phenotype as primary cultured murine microglia (both sources were F4/80+, FcR+, Mac-1+, Mac-2+, Bandeira lectin binding+, and

Characterization of microglial CD40 in vitro

CD40 expression was detected by flow cytometric analysis and found to be constitutively expressed on cultured N9 microglial cells at low levels (Fig. 1A). To investigate whether IFN-γ might enhance microglial CD40, we measured CD40 expression on microglial cells which were treated with IFN-γ (10 U/ml) for 12 h. Results showed a dose-dependent enhancement of CD40 expression on IFN-γ-treated microglial cells (Fig. 1B). Furthermore, this response was specific to IFN-γ, as microglial CD40

Discussion

Our results showed that microglial CD40 expression was markedly enhanced by a low dose of IFN-γ (10 U/ml). In order to determine if CD40 expression on untreated microglia was functional, we ligated microglial CD40 with CD40L and measured TNF-α production. We found that significant levels of TNF-α were produced by CD40-ligated microglia, and TNF-α release was markedly enhanced by pre-treatment of these cells with a low dose of IFN-γ. Having established the functionality of microglial CD40, we

Acknowledgements

The authors are grateful to Mr. and Mrs. Robert Roskamp for their generous support, which helped to make this work possible. We extend our gratitude to Dr. Paola Ricciardi-Castagnoli (Cellular Pharmacology Center, Milano, Italy) for providing the murine microglial cell lines (N9 and N60). We extend our gratitude to Dr. Jean-Yves Bonnefoy (Glaxo Institute for Molecular Biology, Geneva) for providing human recombinant CD40L. We would also like to thank Dr. Shinjie Song for his assistance in

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