Activated microglia (BV-2) facilitation of TNF-α-mediated motor neuron death in vitro

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Abstract

We have studied the interactions between activated microglia and injured motor neurons using an immortalized murine microglial cell line (BV-2) stimulated with either lipopolysaccharide (LPS) (Escherichia coli) or supernatant from serum-deprived motor neurons (NSC-34 cell line). Both stimuli induced BV-2 activation. Although both BV-2 supernatants induced a subsequent increase in NO generation in otherwise healthy NSC-34 cells, only LPS-activated microglial supernatant induced NSC-34 cell death through a TNF-α-dependent pathway. However, we observed a 20-fold increase in the amount of TNF-α required to kill NSC-34 cells in the absence of LPS-activated BV-2 cell supernatant, indicating that microglia secrete factor(s) that facilitate TNF-α-mediated motor neuron death in vitro.

Introduction

Although it is evident that neuronal injury will incite a prominent perineuronal microglial response (Barron et al., 1990), it is less clear how this response is mediated and what factors determine whether the microglia participate in further neuronal injury. In response to a variety of stimuli, microglia transforms from resting ramified microglia into amoeboid activated microglia that are capable of proliferation, migration and phagocytosis. A number of potentially neurotoxic pathways become upregulated, including an increased expression of iNOS, pre-inflammatory cytokines (Giulian, 1993), proteolytic enzymes and active lysosomal proteases (Banati et al., 1993), and a variety of neurotrophic factors (Jensen et al., 2000). This response can be either neurotoxic and promote further neuronal injury or it can be facilitatory and enhance axonal outgrowth and neuronal survival. Determining the nature of the regulatory events governing this seemingly divergent set of microglia functions has been a source of considerable research. In this series of experiments, we addressed the nature of microglial-mediated motor neuron toxicity. We have observed that, in vitro, the microglial response is stimulus-specific and that microglia can respond to injured motor neurons by releasing soluble, TNF-α-independent, factor(s) that induce NOS expression in otherwise healthy motor neurons. Motor neuron death is however dependent on the microglial release of TNF-α.

Section snippets

Cell cultures and experimental conditions

Three different cell lines (BV-2, NSC-34 and L929) were used in the experiments with all experiments performed in triplicate. Statistical analysis was performed on the mean value from the experiments using t-test (SigmaStat; Jandel Scientific, California).

BV-2 cell activation

In response to 1 μg/ml LPS, the BV-2 cells increased NO generation (Fig. 1). When assessed using DAF-2DA fluorescence in the absence of LPS, minimal baseline fluorescence of BV-2 cells was observed (Fig. 1A). In response to activation with 1μg/ml LPS, there was no evidence of increased NO generation at 12 h. However, at 24 h, a marked increase in DAF-2DA fluorescence was observed (Fig. 1B). Both methylisothiourea and PFL inhibited the LPS-induced up-regulation of BV-2 NO generation (Fig. 1E and

Discussion

We have challenged microglia with two different stimuli in the present study and assayed the impact of this on subsequent microglial/motor neuron interactions. LPS has been widely used as an effective stimulator of microglia (Zielasek and Hartung, 1996), but it is relatively nonspecific. The nature of the signal released from degenerating motor neurons (NSC-34 cells) is unknown.

NSC-34 cell serum deprivation, the effect of which is to induce NSC-34 cell death within 48 h, results in the release

Acknowledgements

MJS is supported by the Premiere's Research Excellence Award. Research supported by the London Health Sciences Research Competition Funds and the Scottish Heritage Rite Foundation.

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