S100B secretion is stimulated by IL-1β in glial cultures and hippocampal slices of rats: Likely involvement of MAPK pathway

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Abstract

S100B is an astrocyte-derived cytokine implicated in the IL-1β-triggered cytokine cycle in Alzheimer's disease. However, the secretion of S100B following stimulation by IL-1β has not been directly demonstrated. We investigated S100B secretion in cortical primary astrocyte cultures, C6 glioma cells and acute hippocampal slices exposed to IL-1β. S100B secretion was induced by IL-1β in all preparations, involving MAPK pathway and, apparently, NF-кB signaling. Astrocytes and C6 cells exhibited different sensitivities to IL-1β. These results suggest that IL-1β-induced S100B secretion is a component of the neuroinflammatory response, which would support the involvement of S100B in the genesis of neurodegenerative diseases.

Introduction

Neurodegenerative diseases are commonly associated with chronic inflammatory responses (Eikelenboom et al., 2006). Deposits of protein aggregates (e.g. beta-amyloid) (Wyss-Coray and Mucke, 2002), classical inflammatory activators, and the presence of pro-inflammatory molecules (e.g. cytokines), suggest that neuroinflammation underlies the pathophysiology of these disorders (Hoozemans et al., 2006, Ralay Ranaivo et al., 2006, Simi et al., 2007). However, the full role of neuroinflammation is still controversial, because the inflammatory response is also related to the mechanism of neuroprotection (Marchetti and Abbracchio, 2005).

Astrocytes, together with microglia, are the main cells involved in the inflammatory response in the central nervous system; these cells exhibit an activated phenotype in neurodegenerative diseases, producing several inflammatory mediators and displaying phagocytic activity (Hauwel et al., 2005, Wyss-Coray and Mucke, 2002). Glial activation leads to the expression of the neurotrophic cytokines, interleukin-1 beta (IL-1 β) and S100B (Donato, 2001, Gruden et al., 2007, Liu et al., 2005, Mrak and Griffin, 2005, Sheng et al., 2000). Recent studies have suggested that the expressions of these cytokines play a central role in the development of neuroinflammation, initiating and sustaining a cascade of events that result in progressive neuronal death (Allan et al., 2005, Royston et al., 1999, Simi et al., 2007).

S100B is a calcium-binding protein, predominantly expressed and secreted by astrocytes in vertebrate brain (Marenholz et al., 2004). Intracellularly, S100B binds to many protein targets, possibly modulating cytoskeleton plasticity, cell proliferation and astrocyte energy metabolism (Donato, 2001, Van Eldik and Wainwright, 2003). High levels of brain tissue S100B have been observed in neurodegenerative disorders, including Alzheimer's disease (Griffin et al., 1998). The extracellular effect of S100B, observed in cell cultures, depends on its concentration. At sub-nanomolar levels, S100B stimulates neurite growth and promotes neuronal survival; however, at micromolar levels this molecule produces opposite effects and can even induce neuronal apoptosis, leading to the induction of pro-inflammatory cytokines such as IL-1β, tumor necrosis factor α and inflammatory stress-related enzymes, such as inducible nitric oxide synthase (Hu et al., 1996).

According to the hypothesis of Griffin et al. (1998), IL-1β from activated microglia induces S100B upregulation in astrocytes. In fact, mRNA expression data provides evidence of cross-talk between these proteins, with each upregulating each other (Mrak and Griffin, 2005). However, the increase in S100B mRNA, induced by IL-1β, is not necessarily accompanied by an elevation in S100B protein (Hinkle et al., 1998). C6 glioma cells treated with IL-1β induced an increase in S100B mRNA and protein after 24 h (Sheng et al., 1996). On the other hand, IL-1β decreased S100B mRNA in astrocyte cultures after 48 h and this effect lasted for at least 7 days, although no change in S100B protein was observed during this time (Hinkle et al., 1998). These results may indicate a different response to IL-1β in astrocyte cultures and C6 glioma cells.

In addition, it should be mentioned that, in contrast to IL-1β, not all S100B produced is necessarily exported. Therefore, it is important to appropriately evaluate the secretion of S100B in the presence of IL-1β. However, to our knowledge, no study has directly evaluated IL-1β-induced S100B secretion. In this study, we investigated S100B secretion in rat cortical astrocyte cultures, C6 glioma cells and acute hippocampal slices exposed to IL-1β. We also investigated the involvement of mitogen-activated protein kinase (MAPK) and NF-kappaB in S100B secretion, under this condition.

Section snippets

Materials

Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco BRL. Interleukin-1beta (IL-1β), pyrrolidine dithiocarbamate (PDTC), S100B, monoclonal anti-S100B antibody (SH-B1), 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), dithiothreitol (DTT), orthovanadate, o-phenylenediamine (OPD), flasks and other materials for cell culture were purchased from Sigma; fetal calf serum was from Cultilab (São Paulo, Brazil); peroxidase-conjugated IgGs were from Amersham; Isohelenin, SN-50,

Results

A rapid S100B secretion response (at 15 min) was observed in primary astrocyte cultures exposed to IL-1β (Fig. 1A). Interestingly, very low levels of IL-1β (1–100 pg/mL) induced S100B secretion, but levels of 1–10 ng/mL were not able to modify basal S100B secretion. On the other hand, in C6 glioma cells (Fig. 1B) and hippocampal slices (Fig. 1C), an increase in S100B secretion was observed at 1 h after IL-1β addition at concentration of 1 ng/mL.

The S100B secretion in astrocytes was transitory.

Discussion

Astrocytes and microglia, activated during brain injury, are responsible for the clearance of debris, as well as the secretion of cytokines and neurotrophic factors. However, chronic glial activation, with the release of pro-inflammatory cytokines, eicosanoids and NO, contributes to dysfunction and death neuronal. In fact, IL-1β and the astrocyte-derived cytokine, S100B, have been correlated with neuroinflammation disorders and neurodegenerative diseases (Allan and Rothwell, 2003, Lam et al.,

Acknowledgements

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and FINEP/Rede IBN 01.06.0842-00. We would like to thank Ms. Alessandra Heizelmann for the technical support with cell culture.

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