Elsevier

Brain Research

Volume 1159, 23 July 2007, Pages 8-17
Brain Research

Research Report
The function of microglia, either neuroprotection or neurotoxicity, is determined by the equilibrium among factors released from activated microglia in vitro

https://doi.org/10.1016/j.brainres.2007.04.066Get rights and content

Abstract

Opposing functions of activated microglia, namely neuroprotection or neurotrophy versus neurodestruction or neurotoxicity, have been observed in a number of experimental models of neurotrauma and neurodegenerative diseases. However, the mechanism(s) involved in the determination of which function activated microglia execute under a given set of conditions still remains to be elucidated. Our current in vitro study has revealed that a neuroprotective/neurotrophic or a neurodestructive/neurotoxic microglial function may be configured by the equilibrium among various microglial factors released into the microenvironment. When NSC-34 neurons were treated with lower concentrations of lipopolysaccharide-stimulated BV-2 microglial conditioned medium (LPS-BVCM), viability of the NSC-34 neurons increased, outgrowth of neuronal processes was promoted, and the formation of 2,5-hexanedione-induced aggregates was prevented. However, when NSC-34 neurons were treated with higher concentrations of the same LPS-BVCM, neuronal viability was reduced, apoptosis was induced and outgrowth of neuronal processes was prevented. Measurement of the cytokines tumor necrotic factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in the LPS-BVCM has shown that the upregulation in expression for each cytokine varied both temporally and quantitatively. It is postulated that an alteration in the concentration of the LPS-BVCM might significantly affect the functional balance of microglial factors in the microenvironment with a resultant different microglial function.

Introduction

Microglial cells, distributed ubiquitously throughout the central nervous system (CNS), serve as a pathological sensor and become activated in response to harmful stimuli (Kreutzberg, 1996). Activated microglia serve as scavenger cells by gradually increasing in numbers, migrating towards injury sites or invaded pathogens and transforming from resting ramified cells to phagocytotic amoeboid cells (Kim and de Vellis, 2005). In addition to upregulation of surface receptors or antigens, such as complement receptor type 3 (CR3) and major histocompability complex type II (MHC-II), activated microglia can also release or upregulate a plethora of cytokines, chemokines and enzymes including IL-1β, IL-6, TNF-α, transformation growth factor-β1 (TGF-β1), macrophage-colony stimulating factors (M-CSF) (Hao et al., 2001, Hao et al., 2002), inducible nitric oxide synthase (iNOS) (Brown and Bal-Price, 2003, Minghetti and Levi, 1998), neural growth factor (NGF), neurotrophin-3 (NT-3), and brain neuronal derived factor (BNDF) (Nakajima et al., 2001). It has now been widely accepted that microglia play important roles in homeostasis and pathogenesis in the CNS.

Microglia have been confirmed to possess neurotoxicity. Cytokines, chemokines and enzymes are very important in such microglial function. For instance, IL-1β has been shown to mediate general inflammatory responses that promoted further secretion of pro-inflammatory cytokines (for example IL-6, IL-8, CSF, and TNF-α) (Ehrlich et al., 1998). The recombinant IL-1 receptor antagonist significantly reduced the degree of damage following brain injury (Toulmond and Rothwell, 1995). TNF-α has been found to have a prominent chemotactic effect and be able to induce an increased expression of cell surface antigens and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells and astrocytes (Shrikant et al., 1996). This may be directly related to the observed migration of inflammatory cells into the CNS following a spinal cord injury (Isaksson et al., 1999). TNF-α and other unknown factor(s) released from LPS-activated microglia could not only lead to a significant loss of motoneurons in vitro (He et al., 2002), but also be directly cytotoxic to oligodendrocytes in vitro and induce significant demyelination in vivo (Probert et al., 1995). Upregulation of M-CSF and its receptors in neuron-injury-activated microglia was thought to augment the overall inflammatory response, which could result in further damages to neurons (Hao et al., 2002). Therefore, the inhibition of microglial activation may possibly attenuate neuronal degeneration induced either by ischemia (Giulian and Robertson, 1990) or by excitatory neurotoxin (Coffey et al., 1990).

On the other hand, activated microglia have also been reported to possess neuroprotective/neurotrophic effects in vitro and in vivo. Activated microglia could release IL-1α and IL-6 to protect neuronal cell line from hydrogen peroxide-induced oxidative attack (Bissonnette et al., 2004). The microglial conditioned medium could support the survival of mesencephalic neurons isolated from the embryonic rat (Nagata et al., 1993). The exogenous microglia directly applied to the hippocampus slice cultures could save more neurons after deprivation of oxygen and glucose (Neumann et al., 2006). Transplanted microglial cells were found to facilitate neuronal growth in the injured site in the CNS (Kitamura et al., 2004). Activated microglia could also secrete some neurotrophic factors such as NGF, NT-3 and BNDF which have been demonstrated to be neuroprotective (Nakajima et al., 2001).

Undoubtedly, previous studies have pointed out that microglia possess dual functions (i.e. neuroprotection/neurontrophy and neurondestruction/neurotoxicity) because microglia are able to produce the cytotoxic proinflammatory factors which would induce neuronal cell death as well as neurotrophic factors which could support the survival of neurons. However, the mechanism in determination of which function activated microglia would predominantly execute in various conditions of the CNS still remains to be elucidated.

Based on our previous study that microglial TNF-α requires some other microglial factors to kill neurons (He et al., 2002), we have assumed that the balance between co- and counter-effects of various microglial factors might determine the role of microglia in a given disease or injury condition. In the current study, the dual functions of microglia which have been activated using lipopolysaccharide (LPS) on a motor neuron cell line (NSC-34) have been investigated. The neurotoxin, 2,5-hexanedione (HD), was employed to induce neuropathy in the motor neurons in vitro. The 2,5-HD induced neuropathy was characterized by large axonal swelling associated with multifocal aggregates of neurofilaments in vivo and in vitro, which showed some similarity to certain neurodegenerative diseases (Graham et al., 1995, Hartley et al., 1997, LoPachin et al., 2004). Our current result has indicated that microglial opposing functions, neuroprotection/neurotrophy versus neurondestruction/neurotoxicity, on normal or HD-treated neuronal cells have been related to the concentration of LPS-activated microglial conditioned medium that was applied to neurons.

Section snippets

Quantification of TNF-α, IL-1β and IL-6 in LPS-BVCM by ELISA

The concentrations of TNF-α, IL-1β and IL-6 in the LPS-BVCM were measured using the ELISA kits. The exposure of BV-2 cells to LPS has increased the concentrations of TNF-α, IL-1β and IL-6 in the serum free culture media in a LPS concentration- and treatment time-dependent pattern. The significant upregulation in release of TNF-α (Fig. 1A) or IL-6 (Fig. 1B) was detected at 4 h after 500 ng/ml or 1 μg/ml LPS treatment and reached the highest concentration (6026.92 ± 630.98 pg/ml for TNF-α or 3050.56

Discussion

It has been believed that the microglia is a double-edged sword with dual functions. But it is still a mystery when and how microglia would exert its neuroprotective/neurotrophic or neurodestructive/neurotoxic function.

In the present study, the dual effects of BV-2 microglial cells on NSC-34 cell viabilities were demonstrated to be related to the intensity of LPS stimulation applied to BV-2 microglial cells and the concentration of LPS-BVCM. BV-2 microglial cells were activated by different

Tissue cell cultures

The NSC-34 hybridoma is a well-characterized murine motor neuron cell line that expresses a number of morphological and biological properties of motoneurons including cholinergic function, expression of neurofilaments and generation of action potentials (Cashman et al., 1992, Durham et al., 1993, Marron et al., 2005). The BV-2 immortalized murine microglial cell line has been widely used in various experimental settings. Recently, by using microarray analysis, BV-2 cell line has been further

Acknowledgments

We are grateful to Dr. Maggie M. Sopper at the University of Western Ontario, Canada for her editorial review and Ms. Nicole Suyun Liu and Ms. Jasmin Qian Ru Lim at the National University of Singapore, Singapore for their editorial assistance. This work was supported by a research grant from the National Medical Research Council of Singapore (NMRC/0788/2003).

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