NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β
Introduction
An early and common feature of the majority of brain pathologies is the activation of microglia, a process characterized by a complex series of events. Among these events, migration of microglia to the site of brain injury represents a crucial step for restoring tissue homeostasis (Kreutzberg, 1996, Streit, 2005).
Several studies indicate that microglial chemotaxis and chemokinesis are regulated by secreted factors, including chemokines (such as monocyte chemoattractant protein-1, macrophage colony stimulating factor, MIP-1), complement factors C3a and C5a, growth factors and several cytokines (Ambrosini and Aloisi, 2004, Carter and Dick, 2003, Paglinawan et al., 2003). Damaged neurons as well as neurons surrounding the lesions could also attract and activate glial cells through the release of factors such as purine derivatives (ATP) (Honda et al., 2001, Davalos et al., 2005, Petersen and Dailey, 2004).
Among the growth factors that may influence microglial mobility, we were interested in analysing whether nerve growth factor (NGF) and transforming growth factor-β (TGF-β, two multifunctional peptides that share neuroprotective and anti-inflammatory proprieties (Unsicker and Strelau, 2000, Tabakman et al., 2004) were able to induce and/or modulate the migratory behaviour of microglial cells.
NGF, the first identified member of the neurotrophin family, has been studied most extensively for its essential role in regulating growth, development and function of peripheral sympathetic neurons and central cholinergic neurons (Levi-Montalcini, 1987). In addition to these neuronal populations, glial cells are also targets, as well as sources, of NGF (Mallat et al., 1989, Elkabes et al., 1996, Heese et al., 1998).
Several lines of evidence indicate that NGF, through direct or/and indirect effects on immune competent cells, regulates immune and inflammatory responses. NGF is known to stimulate the survival and differentiation of mast cells, to promote the release of mediators from basophils and macrophages and to be a chemotactic factor for mast cells and macrophages (Bischoff and Dahinden, 1992, Matsuda et al., 1991, Sawada et al., 2000). In addition, the expression of NGF and that of its low affinity receptor p75 have been found up-regulated in specific brain regions of rats with experimental allergic encephalomyelitis (EAE) (De Simone et al., 1996) and immunohistochemical studies have revealed a high expression of TrkA and p75 on both glial and inflammatory cells in chronic active MS lesions (Valdo et al., 2002).
TGF-β is a key regulator of diverse biological processes, including tissue differentiation, cell proliferation and cell migration (Roberts and Sporn, 1993). In normal brain, TGF-β is present at very low concentrations, whereas its expression is strongly increased in activated glial cells in the injured brain (Unsicker and Strelau, 2000, Lehrmann et al., 1995). Increased levels of TGF-β has also been described in several chronic neurodegenerative pathologies such as Alzheimer and Parkinson diseases (Rota et al., 2006, Vagnucci and Li, 2003, Vawter et al., 1996), and TGF-β mRNA was found up-regulated mainly in activated microglial cells, in animal models of prion disease and of focal cerebral ischemia (Lehrmann et al., 1995, Cunningham et al., 2002). TGF-β inhibits microglial production of the proinflammatory molecules IL-1β and TNF-α and the expression of Class I MHC molecules (Kim et al., 2004). In addition, TGF-β has been found to be neuroprotective for hippocampal neurons through the activation of the PI3kinase/Akt and MAPK/ERK 1, 2 pathways (Zhu et al., 2004) and to positively modulate proliferation and differentiation of neural stem cells in adrenalectomized rats (Battista et al., 2006). Along with TGF-β, NGF has been found to influence the proliferation, migration and differentiation of stem cells in the brain of adult rodents with a selective cholinergic lesion in the basal forebrain (Calzà et al., 2003). The release of TGF-β and NGF by microglia is promoted by interaction with apoptotic but not necrotic neurons, suggesting that these two factors may contribute to the silent and safe removal of apoptotic neurons and facilitate the functional recovery of the surrounding compromised neurons (De Simone et al., 2003).
Based on the above observations, the present study was undertaken to understand whether microglial migration is affected by NGF and TGF-β and unravel possible interactions between the two growth factors in regulating this process.
Section snippets
Reagents
All cell culture reagents were from Invitrogen (Grand Island, NY, U.S.A) and virtually endotoxin free (less then 10 E.U./ml as determined by the manufacturer). Growth factors and other treatments used for the microglia stimulation were obtained from the following sources: NGF (murine β-NGF, prepared and purified according to a standard procedure, Bocchini and Angeletti, 1969) was kindly provided by Dr. Luigi Aloe (CNR, Rome, Italy), recombinant human TGF-β1 (R&D System, USA), K252a (Calbiochem,
NGF promotes migration of microglial cells
Microglial cells were challenged with increasing concentrations of NGF (10 ng/ml–1000 ng/ml). As shown in Fig. 1, NGF induced a significant increase in the percentage of migrated cells in a dose dependent manner with a bell-shaped curve. A significant migration was detectable at 10 ng/ml NGF concentration. Maximal migratory response was obtained at 100 ng/ml of NGF and subsequent experiments were performed using this NGF dose. To verify whether the microglial migratory response was due to
Discussion
In the present study, we show that NGF stimulates the migration of rat microglial cells through mechanisms involving the high affinity receptor TrkA and MAPK/ERK kinases. In addition, NGF chemotactic activity is affected in a complex way by TGF-β, a pleiotropic cytokine known to control the process of microglial activation.
NGF exerts its cellular effects through the activities of two different receptors. The low affinity p75 neurotrophin receptor is a member of the TNF-receptor superfamily,
Acknowledgments
We are grateful to Francesca Aloisi for reading and discussing the manuscript; Maurizio Nunziati and Agostino Eusepi are acknowledged for technical support and animal care.
This work has been supported by Istituto Superiore di Sanità, grant no.C3A7 (art.524) to RDS and the Italian Ministry of Education, University and Research (FIRB-MIUR, Grant no. H91 to LM).
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