Dopamine inhibits responses of astroglia-enriched cultures to lipopolysaccharide via a β-adrenoreceptor-mediated mechanism
Introduction
Diverse forms of acute and chronic neurodegeneration, including brain trauma, stroke, Alzheimer's disease, and Parkinson's disease (PD), are accompanied by inflammation in the brain Raine, 1994, McGeer and McGeer, 1995, Allan and Rothwell, 2001, Hirsch et al., 2003. Astrocytes, together with microglia, are the main effectors cells of innate immune responses in the central nervous system (CNS) and can be activated in neurodegenerative diseases to produce an array of inflammatory mediators Eddleston and Mucke, 1993, Dong and Benveniste, 2001. The resulting inflammation may modulate neurodegenerative processes in either a beneficial or a detrimental fashion (Wyss-Coray and Mucke, 2002).
Lipopolysaccharide (LPS), a bacterial endotoxin which is responsible for multiple pathophysiological changes associated with Gram-negative infections, is widely used as a proinflammatory stimulus in a variety of experimental settings (Mukaida et al., 1996). Both in vivo Buttini et al., 1997, Arimoto and Bing, 2003 and in vitro Chung and Benveniste, 1990, Galea et al., 1992, Gayle et al., 2002 studies have demonstrated that LPS challenge induces upregulation in the expression of several inflammatory mediators in glial cells, including nitric oxide synthase (NOS) and tumor necrosis factor-α (TNF-α) which may be involved in the propagation of a variety of neurodegenerative processes Dawson and Dawson, 1998, Venters et al., 2000.
Dopamine (DA), a prominent neurotransmitter in the brain, is known to have an immunomodulatory role outside the CNS, where several peripheral cell types of the immune system, including lymphocytes and macrophages, express DA receptors Santambrogio et al., 1993, Ricci et al., 1994. DA suppresses the production of TNF-α and augments the release of IL-6 in adrenal zona glomerulosa cells (Ritchie et al., 1996) while in mice, DA receptor agonists and antagonists modulate LPS-induced TNF-α levels in plasma and in peritoneal macrophages (Hasko et al., 1996). More recently, it has been reported that DA attenuates LPS-evoked IL-12 p40 production in macrophages (Hasko et al., 2002) as well as nitric oxide synthase (NOS) expression and NO release in C6 glioma cells (Mazzio et al., 2002). While this suggests that DA may exert an immunomodulatory function, to date, there is no information available regarding the modulatory effect of DA on inflammatory responses of astroglial cells.
We here show that, in rat primary cultures enriched in astrocytes, DA inhibits the release of both TNF-α and NO, an effect not affected by selective antagonists of DA receptors nor mimicked by selective DA receptor agonists, but, rather, fully reversed by selective antagonists of β-adrenergic receptors. These data implicate the existence of a functional cross talk between DA and adrenergic receptors in astroglial cells which can modulate inflammatory responses in brain parenchyma.
Section snippets
Reagents and media
Dulbecco's modified Eagle's Medium (DMEM), foetal calf serum, l-glutamine and penicillin/streptomycin were purchased from Biochrom (Berlin, D). Culture dishes and plates were purchased from Iwaki (Japan). ELISA plates (Maxisorp) were purchased from Nunc (Roskilde, DK). RT-PCR reagents were purchased from Promega (Madison, WI). Oligonucleotides were purchased from MWG (Ebersberg, D). All the other reagents, including Lipopolysaccharide from Escherichia coli stereotype O26:B6 (LPS),
LPS induces the expression of TNF-α and NOS2 mRNA and the accumulation of TNF-α protein and nitrite in the culture medium
At different times (0.5, 2, 5, 24 h) after exposure to LPS (1 μg/ml), total mRNA was extracted from cell cultures enriched in astroglia, retrotranscribed and subjected to PCR by using primers specific for the genes of interest. While constitutive TNF-α and inducible NOS (NOS2) mRNA was not detected in untreated cultures, we observed a rapid increase of TNF-α mRNA in response to LPS (1 μg/ml), an effect already maximal at 30 min after LPS exposure. Upregulation of TNF-α mRNA was transient as it
Discussion
Dopaminergic systems have been the focus of much research, primarily because alteration in DA neurotransmission results in major diseases such as PD and schizophrenia. DA has also been shown to act as a modulator of inflammatory responses in several cell lines of the immune system which express DA receptors Ricci et al., 1994, Santambrogio et al., 1993, Hasko et al., 2002. In this study, we investigated the modulatory effects of DA in primary CNS cell cultures responding to inflammatory
Acknowledgments
This work was partly supported by a grant (no. 3933) from the Italian University and Scientific and Technological Research Ministry (MURST). We would like to thank L. Vallo and B. Tubioli for their help in initiating these experiments.
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