Research ReportThe role of TNF-alpha and its receptors in the production of NGF and GDNF by astrocytes
Introduction
Neurotrophic factors (NTFs) regulate survival, functional maintenance, and phenotypic development of neuronal cells. Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are two major neurotrophic factors that play important roles in the development of various neurodegenerative diseases, such as Alzheimer disease and Parkinson's disease (reviewed in Siegel and Chauhan, 2000).
In the central nervous system (CNS), not only neuronal cells but also glial cells produce NTFs. Astrocytes support neuronal cells by secreting NTFs such as NGF, brain-derived neurotrophic factor (BDNF), and GDNF (Brodie, 1996, Brodie et al., 1998, Galve-Roperh et al., 1997, Appel et al., 1997). Therefore, these NTFs and inducers of NTF biosynthesis would be expected to have enormous therapeutic potential for neurodegenerative diseases and traumatic, ischemic, and inflammatory brain lesions. Tumor necrosis factor-α (TNF-α), a major proinflammatory cytokine, is produced by a variety of immune cells, including T and B lymphocytes, natural killer cells, basophils, eosinophils, neutrophils, and monocytic cells. Non-immune cells, including Kupffer cells (Magilavy and Rothstein, 1988), fibroblast (Havell and Sehgal, 1991), smooth muscle cells, epithelial cells, glial cells, and neurons also produce TNF-α(Sawada et al., 1989, Wang et al., 2003). TNF-α binds to two different receptors, TNFR1(p55) and TNFR2(p75), that mediate a wide variety of biological responses to cells bearing these receptors (Wang et al., 2003; reviewed in MacEwan, 2002).
The primary source of TNF-α in the CNS is activated microglia. Astrocytes can also secrete TNF-α when stimulated with LPS, but they produce 10 times less TNF-α than microglia (Sawada et al., 1989, Wood, 1995). During the development of nervous system, TNF-α modulates the cell cycle and metabolism (Mizuno et al., 1994). In pathological conditions, TNF-α reportedly induces demyelination and/or neuronal degeneration either directly or indirectly via the production of other proinflammatory cytokines such as reactive oxygen species (ROS) and nitric oxide (NO) (Selmaj et al., 1991, Tanaka et al., 1994, Meda et al., 1995, Hu et al., 1997). TNF-α also induces the secretion of NTFs including NGF and GFNF (Brodie, 1996, Appel et al., 1997, Hattori et al., 1996).
In this study, we demonstrate that the NGF and GDNF induction by TNF-α in astrocytes is mediated by both TNFR1 and TNFR2. In addition, TNF-α derived from astrocytes works in autocrine manner. As a result, not only exogenous TNF-α but also astrocyte-derived TNF-α contributes to NGF and GDNF production by astrocytes. Therefore, astrocytes may function autonomously to upregulate their neuroprotective function to respond to inflammation in the CNS.
Section snippets
Expression of TNF-α receptors on astrocytes
To confirm the presence of TNF-α receptors on astrocytes, we first examined the expression of TNFR1 and TNFR2. Flow cytometric analysis revealed that astrocytes express both TNFR1 and 2 on their surface (Fig. 1).
TNF-α induces NGF and GDNF production by astrocytes
When stimulated with 1–50 ng/ml TNF-α, astrocytes transcribed mRNA for both NGF and GDNF as determined by quantitative real time PCR (Fig. 2) . Expression of NGF mRNA peaked in 12 h after TNF-α stimulation, while maximum expression of GDNF occurred earlier. The induction of NGF and GDNF
Discussion
Many studies have focused on the role of astrocytes in neurodegenerative diseases and their production of neurotrophic factors as a potential therapy for promoting the survival and function of injured neurons. Recent studies have shown that acute axonal loss occurs in multiple sclerosis (MS), and its animal model, experimental autoimmune encephalomyelitis (EAE) (reviewed in Bjartmar et al., 2003), and that the administration of NGF ameliorates clinical symptoms of EAE (Flugel et al., 2001,
Drugs and antibodies
Recombinant murine TNF-α, monoclonal goat anti-mouse TNF-α IgG antibody, and neutralizing anti-TNFR1 and anti-TNFR2 antibodies were obtained from Genzyme/Techne (Boston, MA). These antibodies have been characterized previously (Sheehan et al., 1995). Escherichia coli lipopolysaccharide (LPS) was purchased from Difco (Detroit, MI).
Cell culture
Astrocyte-enriched cultures were prepared from the primary mixed glial cell cultures of newborn C57BL/6 mice (SLC, Shizuoka, Japan), as described previously (Suzumura
Acknowledgments
This work was supported in part by the Neuroimmunological Disease Research Committee grant from the Ministry of Health, Labour, and Welfare of Japan, and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology. This work was also supported by the 21st COE Program “Integrated Molecular Medicine for Neuronal and Neoplastic Disorders” from the Ministry of Education, Culture, Sports, Science, and Technology.
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