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NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β
Journal of Neuroimmunology 190 (2007) 53 – 60 www.elsevier.com/locate/jneuroim
NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β R. De Simone ⁎, E. Ambrosini, D. Carnevale, M.A. Ajmone-Cat, L. Minghetti Department of Cell Biology and Neurosciences, Section of Degenerative Inflammatory and Neurological Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Received 10 May 2007; received in revised form 27 July 2007; accepted 27 July 2007
Abstract Activation and mobilization of microglia are early events in the majority of brain pathologies. Among the signalling molecules that can affect microglial behaviour, we investigated whether nerve growth factor (NGF) was able to influence microglial motility. We found that NGF induced chemotaxis of microglial cells through the activation of TrkA receptor. In addition, NGF chemotactic activity was increased in the presence of low concentrations (≤0.2 ng/ml) of transforming growth factor-β (TGF-β), which at this concentration showed chemotactic activity per se. On the contrary, NGF-induced microglial migration was reduced in the presence of chemokinetic concentration of TGF-β (≥ 2 ng/ml). Finally, both basal and NGF-induced migratory activity of microglial cells was increased after a long-term exposure of primary mixed glial cultures to 2 ng/ml of TGF-β. Our observations suggest that both NGF and TGF-β contribute to microglial recruitment. The chemotactic activities of these two pleiotropic factors could be particularly relevant during chronic diseases in which recruited microglia remove apoptotic neurons in the absence of a typical inflammatory reaction. © 2007 Elsevier B.V. All rights reserved. Keywords: Microglia; Inflammation; Cell migration; Neuroprotection
1. 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 ⁎ Corresponding author. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 06 49902488; fax: +39 06 4957821. E-mail address: [email protected] (R. De Simone). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.07.020
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 antiinflammatory 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 (LeviMontalcini, 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
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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. 2. Materials and methods 2.1. 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, Germany), Wortmannin (Stressgen, Canada), PD98059 (SIGMA RBI, USA).
2.2. Cell cultures Microglial cultures were purified from 8 day mixed primary glial cultures obtained from the cerebral cortex of 1-day-old rats, as previously described (Levi et al., 1993) and in accordance with the European Communities Council Directive N. 86/609/EEC. Microglial cells, harvested from the mixed primary glial cultures by mild shaking, were resuspended in Basal Eagle's Medium (BME) without serum, supplemented with 2 mM glutamine and 100 μg/ml gentamicin, and immediately used for the chemotaxis assay. Cell viability was greater than 95%, as tested by Tripan Blue exclusion. Immunostaining revealed that cultures consisted of ≥ 99% positive cells for the microglia/macrophage marker ED1. 2.3. Chemotaxis assay Chemotaxis of microglial cells was assessed using Transwell migration chambers (5 μm-pore polycarbonate filters in 24wells Costar, St. Louis, MO), according to previously published procedures (Agresti et al., 2005). In brief, the bottom wells were filled with medium alone or medium containing the substances under study at the indicated concentration. To distinguish between chemotaxis and enhanced random motility (chemokinesis), in parallel experiments, an equal concentration of the growth factors was added to the upper and lower compartments, thereby neutralizing the chemotactic gradients. Microglial cells were plated into the top wells (6 × 104 cells/well) in BME without serum, and incubated for 4 h at 37 °C. Migrated cells were counted on the lower surface of the filters after fixation in 100% methanol for 3 min and hematoxylin and eosin (H&E) staining. Cells on the top side of the filter were wiped off using a cotton bud. Filters were then removed from the inserts and mounted on glass slides with the stained cells on the upper side, and the cells were counted under a light microscope (16 field were examined for each condition). The number of migrated cells in the presence of NGF and TGF-β was given as % of cell migrated in control condition, taken as 100%. 2.4. Cell viability tests The reduction of cellular 3-(4,5-dimethyl thiazol-2-y1)-2,5diphenyl tetrazolium bromide (MTT) was measured as previously described (Agresti et al., 1996). Microglial cell were incubated with K252a or wortmannin for 4 h and MTT was added during the final 2.5 h of incubation. The medium was then removed and 100 μl of DMSO was added to each well to dissolve the dark blue crystals. The plates were then read on GDV MPT microplate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. 2.5. Determination of Akt and ERK phosphorylation The levels of total and phosphorylated Akt and ERK kinases were measured by a sensitive Fast Activated Cell-based ELISA (FACE TM, Active Motif) kit, designed to monitor proteins activated by phosphorylation, following the instructions
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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 chemotaxis or random activation (chemokinesis), the assay was performed in the presence of equal concentrations of NGF in the upper and lower chambers, thereby neutralizing the chemical gradient. In this condition, microglial migration was strongly prevented indicating that NGF acted as chemoattractant agent. In addition, heat-inactivation of NGF totally abrogated the migration of microglial cells (Fig. 1).
Fig. 1. Migration of microglial cells in response to NGF. Cells were exposed to different concentrations of NGF and incubated for 4 h at 37 °C. NGF was added to the lower compartment of the chemotaxis chamber, to both upper and lower compartments (up/low) or to the lower compartment after heat inactivation. The number of migrated cells in the control and in response to 100 ng/ml NGF was 105 ± 3.8 and 155± 13 cells/mm2. Data are expressed as the % of increase over the control condition, taken as 100%. Data are presented as mean ± SEM (n = 4–12 for NGF dose response; n = 4 for up/low and n = 3 for NGF h.i. of independent experiments run in duplicate). Migration in the presence of 10 or 100 ng/ml was significantly different vs control cultures (p b 0.0005). ⁎p b 0.05; ⁎⁎p b 0.0005.
provided by the manufacturer. Briefly, microglial cells were cultured in 96 well plates at the density of 1.25 × 105 cell/cm2 and maintained for 1 h in BME serum free medium. The cultures were then stimulated with or without NGF (100 ng/ml) for the indicated times. At the end of the treatment, the cells were fixed in 4% of formaldehyde in PBS at room temperature. Formaldehyde solution was then removed and the cells were washed with the PBS containing 0.1% Triton. After for 20 min of incubation at room temperature in quenching buffer (wash buffer containing 1% H2O2 and 0.1% Azide) cell were incubated with the provided anti-phospho Akt and ERK or anti-total antibodies and revealed with HRP-conjugated secondary antibodies and developing solution that provide quantitative and reproducible colorimetric readouts. The phosphorylated Akt and ERK signals were normalized for the number of cells in each well, determined by Crystal violet dye.
3.2. NGF induces microglial migration by the high affinity receptor TrkA NGF exerts its biological actions through two receptors, the low affinity, 75-kD glycoprotein p75, and the high affinity receptor TrkA, a 140-kD molecule with a transmembrane tyrosine kinase domain. The binding of NGF to TrkA triggers its autophosphorylation and initiates multiple second messenger signalling pathways including several kinase cascades (Chao et al., 2006). To examine whether the high affinity receptor TrkA was involved in NGF induced-migration of microglial cells, the protein tyrosine kinase inhibitor K252a was added to the cells at the beginning of the chemotaxis assay. A concentration of 30 nM K252a was shown to be selective for inhibiting the NGF-induced migration of endothelial cells (Dollé et al., 2005). In preliminary experiments, the selectivity and cell toxicity of K252a was tested in the concentration range of 0.1–100 nM. We found that the inhibition of NGF-induced cell migration was already evident at 0.1 nM K252a and that cell vitality checked by the MTT test, was approximately 93% of control cultures at the doses of 0.1 and 1 nM K252a and 77% at 100 nM, a dose not used in our chemotactic assay to avoid
2.6. Statistical analysis Data are expressed as means ± SEM of (n) independent experiments (run in duplicate). Comparison between treatment groups was made by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons. A two tailed probability of less than 5% (i.e. p b 0.05) was taken as statistically significant. 3. Results 3.1. 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
Fig. 2. Effects of the protein tyrosine kinase inhibitor K252a on NGF-induced migration of microglial cells. Cells were stimulated with 100 ng/ml NGF in the presence or absence of K252a at two concentrations (0.1 and 1 nM), as described in Fig. 1. The highest concentration of K252a was also tested in the absence of NGF. Data are presented as mean ± SEM of n = 3 independent experiments run in duplicate. ⁎p b 0.05; ⁎⁎p b 0.0005.
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Fig. 3. Effects of the MAPK/ERK kinase inhibitor PD98059 on NGF-induced migration and effect of NGF stimulation on the MAPK/ERK 1/2 and Akt phosphorylation. A. Cells were incubated with 100 ng/ml NGF, 25 μM PD98059, or with their combination, as described in Fig. 1. Data are presented as mean ± SEM of n = 4 independent experiments run in duplicate. ⁎p b 0.05. B and C. Microglial cells were stimulated with NGF (100 ng/ml) for the indicated times. Phosphorylated and total ERK (B) and Akt (C) were quantified by the Fast Activated Cell-Based ELISA kit. The y axis indicates the ratio between phosphorylated ERK or Akt signals normalized for the number of cells in each well. Data are expressed as mean ± SEM of n = 3 independent experiments.
any unspecific inhibition. At the concentrations of 0.1 and 1 nM, K252a significantly reduced the chemotactic response of microglial cells evoked by 100 ng/ml NGF (Fig. 2), indicating that TrkA signal transduction is essential for NGF chemotactic activity on microglial cells. When administered alone, the inhibitor did not influence the basal migratory response of the cells. The pathways downstream to TrkA were investigated using specific inhibitors for kinases such as MAPK/ERK and PI3K. The presence of the specific MAPK/ERK inhibitor PD98059 (25 μM) induced a slight reduction in the basal migratory activity, and totally prevented that evoked by 100 ng/ml NGF (Fig. 3A), indicating the requirement of pathways mediated by MAPK/ERK for NGF-induced migration. However, the ERK1 and ERK2, which are generally rapidly activated after NGF stimulation in several cell types, were not phosphorylated and, hence, activated upon exposure to 100 ng/ml NGF for 3, 5 or 15 min (Fig. 3B). On the other hand, the specific PI3K inhibitor wortmannin (tested at the non-toxic concentration of 1 μM), completely blocked the migratory activity of microglial cells, regardless the presence or absence of NGF (data not shown), preventing us from any conclusion on PI3K involvement in NGF-induced chemotaxis. When we measured the level of phosphorylation of Akt, a PI3K downstream effector, we found that 100 ng/ml NGF exposure for 3, 5 or 15 min, did not influence the phosphorylation state of Akt (Fig. 3C), similarly to what observed for the ERK1/2 kinases. It is important to note that significant levels of Akt phosphorylation were detectable in control conditions.
observed at 0.0005 ng/ml TGF-β. The percentage of migrated cells was substantially similar over a broad range of concentration (0.0005–20 ng/ml TGF-β although there was a trend to decrease at the highest concentration tested (127% ± 7.7 for 20 ng/ml TGF-β). The presence of TGF-β in both the upper and lower compartments at the different doses revealed that the migratory response induced by 2 ng/ml TGF-β was largely due to chemokinesis (Fig. 4). According to what observed in murine mast cells (Gruber et al., 1994), at lower concentrations (0.0005 and 0.2 ng/ml), the activity of TGF-β on microglial motility was exclusively chemotactic, since the presence of the cytokine in both compartments completely inhibited the migration (Fig. 4). In the presence of heat-denatured TGF-β we did not observe any significant migratory response (data not shown).
3.3. Modulation of NGF-induced microglial cell migration by TGF-β
Fig. 4. Migration of microglial cells in response to TGF-β. Cells were exposed to different concentrations of TGF-β and incubated for 4 h at 37 °C. TGF-β was added to the lower chemotaxis compartments (lower) or to both compartments (up/low). Data are presented as mean ± SEM of n = 3–9 independent experiments run in duplicate, with the exception of TGF-β 0.0005 and 0.2 ng/ml up/low, n = 3, not included in the ANOVA analysis. ⁎p b 0.05 and ⁎⁎p b 0.005 vs control cultures.
We first analysed the activity of TGF-β by exposing microglial cells to increasing concentrations of the cytokine. As shown in Fig. 4, a significant migratory response was already
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Fig. 5. Modulation of NGF-induced migration by TGF-β. A. Cells were treated with TGF-β (0.0005 ng/ml) and NGF (10 ng/ml) alone or in combination for 4 h at 37 °C. Data are expressed as mean ± SEM of n = 4 independent experiments run in duplicate. ⁎p b 0.05. B. Cells were stimulated with TGF-β (2 ng/ml) and NGF (10 ng/ml) alone or in combination for 4 h at 37 °C. Data are presented as mean ± SEM of n = 4 independent experiments. ⁎p b 0.05.
The interaction between NGF and TGF-β was then investigated. To analyse the possible additive or synergistic effects of the two growth factors, we used TGF-β concentrations generating chemotactic or chemokinetic migratory responses (0.0005 ng/ml TGF-β and 2 ng/ml, respectively). In the presence of a suboptimal concentration of NGF (10 ng/ml), 0.0005 ng/ml TGF-β increased the migratory response of microglia, although the effect reached statistical significance only when compared to TGF-β alone (Fig. 5A). On the contrary, the percentage of migrated cells induced by 10 ng/ml NGF was significantly reduced when combined with 2 ng/ml TGF-β (Fig. 5B).
These findings suggest that the nature of the interactions between the two factors is determined by the local concentration of TGF-β, being the effects on microglial migration additive in the presence of low TGF-β concentration and counteractive at higher concentration. To further explore the activities of the two factors on microglial motility, we analysed the effect of a long term exposure to chemokinetics concentration of TGF-β on the NGF-induced microglial migration. To this aim, primary mixed glial cultures were exposed to TGF-β (2 ng/ml) for 16 h. After this time, microglial cells were harvested and chemotaxis assay performed in the presence of the optimal concentration of NGF (100 ng/ml).
Fig. 6. Effect of long-term exposure to TGF-β on microglial migration. Microglial cells were exposed overnight (o.n.) to TGF-β (2 ng/ml) and then harvested and tested for their migratory behaviour. Cells from control and TGF-β-o.n. treated cultures were exposed to 100 ng/ml NGF. A. Data are expressed as % of migration in control cultures and are presented as mean ± SEM of n = 3 independent experiments, run in duplicate. TGF-β pretreatment increased significantly the migratory activity in basal conditions and when stimulated with 100 ng/ml NGF. ⁎p b 0.05. B. Representative photomicrographs of microglial migration in the chemotactic chamber: a, control medium; b, NGF (100 ng/ml); c, TGF-β (2 ng/ml); d, TGF-β o.n. (2 ng/ml) + NGF (100 ng/ml).
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TGF-β pre-treatment significantly increased both basal and NGFinduced migratory activity of microglial cells (Fig. 6A and B). 4. 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, which binds also other neurotrophins such as BDNF, NT-4 and NT-3 (Chao et al., 2006). The high affinity TrkA receptor, is a glycoprotein belonging to the tyrosine kinase-receptor family and specifically binds NGF (Chao et al., 2006). The engagement of TrkA receptor rapidly elicits the autophosphorylation of the receptor on tyrosine residues and, consequently, multiple signal transduction pathways including those mediated by kinases such as MAPK/ERK and PI3K (Arevalo and Wu, 2006). Activation of these kinases is involved in many neuronal functions, including cell survival, differentiation, synapse formation and plasticity, and migration of neurons elicited by neurotrophins (Behar et al., 1997; Ho et al., 2005). Recent studies have reported that MAPK/ERK and PI3K/Akt pathways are involved in the NGF-induced migration of mast cells and endothelial cells (Sawada et al., 2000; Rahbek et al., 2005). In line with these studies, we found that NGF-induced chemotaxis of microglial cells was prevented by specific inhibitors of TrkA kinase activity or MAPK/ERK cascade. However, brief stimulation of microglial cells with NGF (3, 5 or 15 min) did not modify the levels of ERK 1,2 phosphorylation. On the other hand, our data cannot be conclusive on the involvement of PI3K/Akt pathway since wortmannin, a specific inhibitor of PI3K, prevented both spontaneous and NGF-induced microglial migration and NGF did not modify the phosphorylation state of Akt. Thus, the signals downstream to TrkA and MAPK/ERK mediating NGF-induced microglial migration remain to be identified. We also found that TGF-β depending on the concentrations used, exerts chemotactic or chemokinetic effects on microglial cells. At the concentration of 2 ng/ml, TGF-β predominantly increased microglial cell motility and, when combined with NGF, it decreased microglial cell chemotaxis. On the contrary, at lower concentrations (0.0005–0.2 ng/ml) TGF-β acted as chemotactic factor and significantly enhanced the migratory response elicited by NGF. Interestingly, a prolonged exposure of primary mixed glial cultures to chemokinetic concentrations of TGF-β (2 ng/ml) remarkably increased the spontaneous motility of microglial cells as well as that induced by NGF. This last phenomenon could be due either to a direct effect of TGF-β on the cell machinery involved in the motility of microglial cells, or to indirect effects mediated by factors released by microglia or astrocytes during TGF-β pre-treatment of mixed cultures, which in turn may influence the migratory behaviour of these cells.
In virtually all brain pathologies, including ischemia, trauma or chronic inflammatory processes, microglia release and/or respond to several pro- and anti-inflammatory cytokines, chemokines and growth factors (Minghetti, 2005; Mrak and Griffin, 2005; Hauwel et al., 2005). Because of the high redundancy, the diversity of their actions, the antagonism or synergism among all these molecules, the role of each factor in brain damage or recovery is difficult to be established. NGF and TGF-β expression has been shown to increase in response to CNS injury or inflammation and it has been hypothesized that these factors may contribute to the resolution of tissue inflammation (Hohlfeld et al., 2006; Boche et al., 2006). TGF-β expression is increased during the recovery phase of EAE (Issazadeh et al., 1998) and TGF-β-treated microglia induce oligodendrocyte precursor cell migration (Lalive et al., 2005). Along with TGF-β, NGF production is increased in a variety of CNS disorders, including brain injury (Kossmann et al., 1996; Brown et al., 2004; DeKosky et al., 2004), EAE (De Simone et al., 1996), and multiple sclerosis relapsing phase (Laudiero et al., 1992). In addition, NGF, administered or delivered by transgenic T cells, delays clinical EAE onset in the marmoset and mice (Villoslada et al., 2000; Flugel et al., 2001). Since all these pathological conditions are associated with the presence of cytokines that stimulate the production of NGF by astrocytes, it has been hypothesized that NGF can act as an autocrine anti-inflammatory cytokine produced in the brain. These observations, along with the present results, lead us to hypothesize that the presence of NGF and TGF-β at sites of injury, where inflammatory cells as well as dying neurons are present (Lindholm et al., 1987; Crutcher et al., 1993), may promote microglial recruitment to the core of the lesion. Once recruited, microglial cells can be induced by these factors to acquire the anti-inflammatory and protective phenotype necessary to the efficient and silent removal of apoptotic bodies. In line with this hypothesis, we have shown that the specific interaction of microglial cells with apoptotic neurons attenuates the release of pro-inflammatory cytokines while promoting that of TGF-β and NGF (De Simone et al., 2003). It is possible that, through paracrine mechanisms, NGF and/or TGF-β may further recruit microglial cells to restore tissue homeostasis. Although other factors, including chemokines such as MCP-1, are abundantly released during inflammatory and immune-mediated diseases, and act as potent microglial chemoattractants (Ambrosini and Aloisi, 2004), the NGF/TGF-β induced migration of microglial cells may become relevant in other settings, characterized by the presence of neuronal apoptotic death such as during brain development or chronic diseases. 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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NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β R. De Simone ⁎, E. Ambrosini, D. Carnevale, M.A. Ajmone-Cat, L. Minghetti Department of Cell Biology and Neurosciences, Section of Degenerative Inflammatory and Neurological Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy Received 10 May 2007; received in revised form 27 July 2007; accepted 27 July 2007
Abstract Activation and mobilization of microglia are early events in the majority of brain pathologies. Among the signalling molecules that can affect microglial behaviour, we investigated whether nerve growth factor (NGF) was able to influence microglial motility. We found that NGF induced chemotaxis of microglial cells through the activation of TrkA receptor. In addition, NGF chemotactic activity was increased in the presence of low concentrations (≤0.2 ng/ml) of transforming growth factor-β (TGF-β), which at this concentration showed chemotactic activity per se. On the contrary, NGF-induced microglial migration was reduced in the presence of chemokinetic concentration of TGF-β (≥ 2 ng/ml). Finally, both basal and NGF-induced migratory activity of microglial cells was increased after a long-term exposure of primary mixed glial cultures to 2 ng/ml of TGF-β. Our observations suggest that both NGF and TGF-β contribute to microglial recruitment. The chemotactic activities of these two pleiotropic factors could be particularly relevant during chronic diseases in which recruited microglia remove apoptotic neurons in the absence of a typical inflammatory reaction. © 2007 Elsevier B.V. All rights reserved. Keywords: Microglia; Inflammation; Cell migration; Neuroprotection
1. 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 ⁎ Corresponding author. Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 06 49902488; fax: +39 06 4957821. E-mail address: [email protected] (R. De Simone). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.07.020
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 antiinflammatory 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 (LeviMontalcini, 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
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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. 2. Materials and methods 2.1. 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, Germany), Wortmannin (Stressgen, Canada), PD98059 (SIGMA RBI, USA).
2.2. Cell cultures Microglial cultures were purified from 8 day mixed primary glial cultures obtained from the cerebral cortex of 1-day-old rats, as previously described (Levi et al., 1993) and in accordance with the European Communities Council Directive N. 86/609/EEC. Microglial cells, harvested from the mixed primary glial cultures by mild shaking, were resuspended in Basal Eagle's Medium (BME) without serum, supplemented with 2 mM glutamine and 100 μg/ml gentamicin, and immediately used for the chemotaxis assay. Cell viability was greater than 95%, as tested by Tripan Blue exclusion. Immunostaining revealed that cultures consisted of ≥ 99% positive cells for the microglia/macrophage marker ED1. 2.3. Chemotaxis assay Chemotaxis of microglial cells was assessed using Transwell migration chambers (5 μm-pore polycarbonate filters in 24wells Costar, St. Louis, MO), according to previously published procedures (Agresti et al., 2005). In brief, the bottom wells were filled with medium alone or medium containing the substances under study at the indicated concentration. To distinguish between chemotaxis and enhanced random motility (chemokinesis), in parallel experiments, an equal concentration of the growth factors was added to the upper and lower compartments, thereby neutralizing the chemotactic gradients. Microglial cells were plated into the top wells (6 × 104 cells/well) in BME without serum, and incubated for 4 h at 37 °C. Migrated cells were counted on the lower surface of the filters after fixation in 100% methanol for 3 min and hematoxylin and eosin (H&E) staining. Cells on the top side of the filter were wiped off using a cotton bud. Filters were then removed from the inserts and mounted on glass slides with the stained cells on the upper side, and the cells were counted under a light microscope (16 field were examined for each condition). The number of migrated cells in the presence of NGF and TGF-β was given as % of cell migrated in control condition, taken as 100%. 2.4. Cell viability tests The reduction of cellular 3-(4,5-dimethyl thiazol-2-y1)-2,5diphenyl tetrazolium bromide (MTT) was measured as previously described (Agresti et al., 1996). Microglial cell were incubated with K252a or wortmannin for 4 h and MTT was added during the final 2.5 h of incubation. The medium was then removed and 100 μl of DMSO was added to each well to dissolve the dark blue crystals. The plates were then read on GDV MPT microplate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. 2.5. Determination of Akt and ERK phosphorylation The levels of total and phosphorylated Akt and ERK kinases were measured by a sensitive Fast Activated Cell-based ELISA (FACE TM, Active Motif) kit, designed to monitor proteins activated by phosphorylation, following the instructions
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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 chemotaxis or random activation (chemokinesis), the assay was performed in the presence of equal concentrations of NGF in the upper and lower chambers, thereby neutralizing the chemical gradient. In this condition, microglial migration was strongly prevented indicating that NGF acted as chemoattractant agent. In addition, heat-inactivation of NGF totally abrogated the migration of microglial cells (Fig. 1).
Fig. 1. Migration of microglial cells in response to NGF. Cells were exposed to different concentrations of NGF and incubated for 4 h at 37 °C. NGF was added to the lower compartment of the chemotaxis chamber, to both upper and lower compartments (up/low) or to the lower compartment after heat inactivation. The number of migrated cells in the control and in response to 100 ng/ml NGF was 105 ± 3.8 and 155± 13 cells/mm2. Data are expressed as the % of increase over the control condition, taken as 100%. Data are presented as mean ± SEM (n = 4–12 for NGF dose response; n = 4 for up/low and n = 3 for NGF h.i. of independent experiments run in duplicate). Migration in the presence of 10 or 100 ng/ml was significantly different vs control cultures (p b 0.0005). ⁎p b 0.05; ⁎⁎p b 0.0005.
provided by the manufacturer. Briefly, microglial cells were cultured in 96 well plates at the density of 1.25 × 105 cell/cm2 and maintained for 1 h in BME serum free medium. The cultures were then stimulated with or without NGF (100 ng/ml) for the indicated times. At the end of the treatment, the cells were fixed in 4% of formaldehyde in PBS at room temperature. Formaldehyde solution was then removed and the cells were washed with the PBS containing 0.1% Triton. After for 20 min of incubation at room temperature in quenching buffer (wash buffer containing 1% H2O2 and 0.1% Azide) cell were incubated with the provided anti-phospho Akt and ERK or anti-total antibodies and revealed with HRP-conjugated secondary antibodies and developing solution that provide quantitative and reproducible colorimetric readouts. The phosphorylated Akt and ERK signals were normalized for the number of cells in each well, determined by Crystal violet dye.
3.2. NGF induces microglial migration by the high affinity receptor TrkA NGF exerts its biological actions through two receptors, the low affinity, 75-kD glycoprotein p75, and the high affinity receptor TrkA, a 140-kD molecule with a transmembrane tyrosine kinase domain. The binding of NGF to TrkA triggers its autophosphorylation and initiates multiple second messenger signalling pathways including several kinase cascades (Chao et al., 2006). To examine whether the high affinity receptor TrkA was involved in NGF induced-migration of microglial cells, the protein tyrosine kinase inhibitor K252a was added to the cells at the beginning of the chemotaxis assay. A concentration of 30 nM K252a was shown to be selective for inhibiting the NGF-induced migration of endothelial cells (Dollé et al., 2005). In preliminary experiments, the selectivity and cell toxicity of K252a was tested in the concentration range of 0.1–100 nM. We found that the inhibition of NGF-induced cell migration was already evident at 0.1 nM K252a and that cell vitality checked by the MTT test, was approximately 93% of control cultures at the doses of 0.1 and 1 nM K252a and 77% at 100 nM, a dose not used in our chemotactic assay to avoid
2.6. Statistical analysis Data are expressed as means ± SEM of (n) independent experiments (run in duplicate). Comparison between treatment groups was made by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons. A two tailed probability of less than 5% (i.e. p b 0.05) was taken as statistically significant. 3. Results 3.1. 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
Fig. 2. Effects of the protein tyrosine kinase inhibitor K252a on NGF-induced migration of microglial cells. Cells were stimulated with 100 ng/ml NGF in the presence or absence of K252a at two concentrations (0.1 and 1 nM), as described in Fig. 1. The highest concentration of K252a was also tested in the absence of NGF. Data are presented as mean ± SEM of n = 3 independent experiments run in duplicate. ⁎p b 0.05; ⁎⁎p b 0.0005.
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Fig. 3. Effects of the MAPK/ERK kinase inhibitor PD98059 on NGF-induced migration and effect of NGF stimulation on the MAPK/ERK 1/2 and Akt phosphorylation. A. Cells were incubated with 100 ng/ml NGF, 25 μM PD98059, or with their combination, as described in Fig. 1. Data are presented as mean ± SEM of n = 4 independent experiments run in duplicate. ⁎p b 0.05. B and C. Microglial cells were stimulated with NGF (100 ng/ml) for the indicated times. Phosphorylated and total ERK (B) and Akt (C) were quantified by the Fast Activated Cell-Based ELISA kit. The y axis indicates the ratio between phosphorylated ERK or Akt signals normalized for the number of cells in each well. Data are expressed as mean ± SEM of n = 3 independent experiments.
any unspecific inhibition. At the concentrations of 0.1 and 1 nM, K252a significantly reduced the chemotactic response of microglial cells evoked by 100 ng/ml NGF (Fig. 2), indicating that TrkA signal transduction is essential for NGF chemotactic activity on microglial cells. When administered alone, the inhibitor did not influence the basal migratory response of the cells. The pathways downstream to TrkA were investigated using specific inhibitors for kinases such as MAPK/ERK and PI3K. The presence of the specific MAPK/ERK inhibitor PD98059 (25 μM) induced a slight reduction in the basal migratory activity, and totally prevented that evoked by 100 ng/ml NGF (Fig. 3A), indicating the requirement of pathways mediated by MAPK/ERK for NGF-induced migration. However, the ERK1 and ERK2, which are generally rapidly activated after NGF stimulation in several cell types, were not phosphorylated and, hence, activated upon exposure to 100 ng/ml NGF for 3, 5 or 15 min (Fig. 3B). On the other hand, the specific PI3K inhibitor wortmannin (tested at the non-toxic concentration of 1 μM), completely blocked the migratory activity of microglial cells, regardless the presence or absence of NGF (data not shown), preventing us from any conclusion on PI3K involvement in NGF-induced chemotaxis. When we measured the level of phosphorylation of Akt, a PI3K downstream effector, we found that 100 ng/ml NGF exposure for 3, 5 or 15 min, did not influence the phosphorylation state of Akt (Fig. 3C), similarly to what observed for the ERK1/2 kinases. It is important to note that significant levels of Akt phosphorylation were detectable in control conditions.
observed at 0.0005 ng/ml TGF-β. The percentage of migrated cells was substantially similar over a broad range of concentration (0.0005–20 ng/ml TGF-β although there was a trend to decrease at the highest concentration tested (127% ± 7.7 for 20 ng/ml TGF-β). The presence of TGF-β in both the upper and lower compartments at the different doses revealed that the migratory response induced by 2 ng/ml TGF-β was largely due to chemokinesis (Fig. 4). According to what observed in murine mast cells (Gruber et al., 1994), at lower concentrations (0.0005 and 0.2 ng/ml), the activity of TGF-β on microglial motility was exclusively chemotactic, since the presence of the cytokine in both compartments completely inhibited the migration (Fig. 4). In the presence of heat-denatured TGF-β we did not observe any significant migratory response (data not shown).
3.3. Modulation of NGF-induced microglial cell migration by TGF-β
Fig. 4. Migration of microglial cells in response to TGF-β. Cells were exposed to different concentrations of TGF-β and incubated for 4 h at 37 °C. TGF-β was added to the lower chemotaxis compartments (lower) or to both compartments (up/low). Data are presented as mean ± SEM of n = 3–9 independent experiments run in duplicate, with the exception of TGF-β 0.0005 and 0.2 ng/ml up/low, n = 3, not included in the ANOVA analysis. ⁎p b 0.05 and ⁎⁎p b 0.005 vs control cultures.
We first analysed the activity of TGF-β by exposing microglial cells to increasing concentrations of the cytokine. As shown in Fig. 4, a significant migratory response was already
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Fig. 5. Modulation of NGF-induced migration by TGF-β. A. Cells were treated with TGF-β (0.0005 ng/ml) and NGF (10 ng/ml) alone or in combination for 4 h at 37 °C. Data are expressed as mean ± SEM of n = 4 independent experiments run in duplicate. ⁎p b 0.05. B. Cells were stimulated with TGF-β (2 ng/ml) and NGF (10 ng/ml) alone or in combination for 4 h at 37 °C. Data are presented as mean ± SEM of n = 4 independent experiments. ⁎p b 0.05.
The interaction between NGF and TGF-β was then investigated. To analyse the possible additive or synergistic effects of the two growth factors, we used TGF-β concentrations generating chemotactic or chemokinetic migratory responses (0.0005 ng/ml TGF-β and 2 ng/ml, respectively). In the presence of a suboptimal concentration of NGF (10 ng/ml), 0.0005 ng/ml TGF-β increased the migratory response of microglia, although the effect reached statistical significance only when compared to TGF-β alone (Fig. 5A). On the contrary, the percentage of migrated cells induced by 10 ng/ml NGF was significantly reduced when combined with 2 ng/ml TGF-β (Fig. 5B).
These findings suggest that the nature of the interactions between the two factors is determined by the local concentration of TGF-β, being the effects on microglial migration additive in the presence of low TGF-β concentration and counteractive at higher concentration. To further explore the activities of the two factors on microglial motility, we analysed the effect of a long term exposure to chemokinetics concentration of TGF-β on the NGF-induced microglial migration. To this aim, primary mixed glial cultures were exposed to TGF-β (2 ng/ml) for 16 h. After this time, microglial cells were harvested and chemotaxis assay performed in the presence of the optimal concentration of NGF (100 ng/ml).
Fig. 6. Effect of long-term exposure to TGF-β on microglial migration. Microglial cells were exposed overnight (o.n.) to TGF-β (2 ng/ml) and then harvested and tested for their migratory behaviour. Cells from control and TGF-β-o.n. treated cultures were exposed to 100 ng/ml NGF. A. Data are expressed as % of migration in control cultures and are presented as mean ± SEM of n = 3 independent experiments, run in duplicate. TGF-β pretreatment increased significantly the migratory activity in basal conditions and when stimulated with 100 ng/ml NGF. ⁎p b 0.05. B. Representative photomicrographs of microglial migration in the chemotactic chamber: a, control medium; b, NGF (100 ng/ml); c, TGF-β (2 ng/ml); d, TGF-β o.n. (2 ng/ml) + NGF (100 ng/ml).
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TGF-β pre-treatment significantly increased both basal and NGFinduced migratory activity of microglial cells (Fig. 6A and B). 4. 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, which binds also other neurotrophins such as BDNF, NT-4 and NT-3 (Chao et al., 2006). The high affinity TrkA receptor, is a glycoprotein belonging to the tyrosine kinase-receptor family and specifically binds NGF (Chao et al., 2006). The engagement of TrkA receptor rapidly elicits the autophosphorylation of the receptor on tyrosine residues and, consequently, multiple signal transduction pathways including those mediated by kinases such as MAPK/ERK and PI3K (Arevalo and Wu, 2006). Activation of these kinases is involved in many neuronal functions, including cell survival, differentiation, synapse formation and plasticity, and migration of neurons elicited by neurotrophins (Behar et al., 1997; Ho et al., 2005). Recent studies have reported that MAPK/ERK and PI3K/Akt pathways are involved in the NGF-induced migration of mast cells and endothelial cells (Sawada et al., 2000; Rahbek et al., 2005). In line with these studies, we found that NGF-induced chemotaxis of microglial cells was prevented by specific inhibitors of TrkA kinase activity or MAPK/ERK cascade. However, brief stimulation of microglial cells with NGF (3, 5 or 15 min) did not modify the levels of ERK 1,2 phosphorylation. On the other hand, our data cannot be conclusive on the involvement of PI3K/Akt pathway since wortmannin, a specific inhibitor of PI3K, prevented both spontaneous and NGF-induced microglial migration and NGF did not modify the phosphorylation state of Akt. Thus, the signals downstream to TrkA and MAPK/ERK mediating NGF-induced microglial migration remain to be identified. We also found that TGF-β depending on the concentrations used, exerts chemotactic or chemokinetic effects on microglial cells. At the concentration of 2 ng/ml, TGF-β predominantly increased microglial cell motility and, when combined with NGF, it decreased microglial cell chemotaxis. On the contrary, at lower concentrations (0.0005–0.2 ng/ml) TGF-β acted as chemotactic factor and significantly enhanced the migratory response elicited by NGF. Interestingly, a prolonged exposure of primary mixed glial cultures to chemokinetic concentrations of TGF-β (2 ng/ml) remarkably increased the spontaneous motility of microglial cells as well as that induced by NGF. This last phenomenon could be due either to a direct effect of TGF-β on the cell machinery involved in the motility of microglial cells, or to indirect effects mediated by factors released by microglia or astrocytes during TGF-β pre-treatment of mixed cultures, which in turn may influence the migratory behaviour of these cells.
In virtually all brain pathologies, including ischemia, trauma or chronic inflammatory processes, microglia release and/or respond to several pro- and anti-inflammatory cytokines, chemokines and growth factors (Minghetti, 2005; Mrak and Griffin, 2005; Hauwel et al., 2005). Because of the high redundancy, the diversity of their actions, the antagonism or synergism among all these molecules, the role of each factor in brain damage or recovery is difficult to be established. NGF and TGF-β expression has been shown to increase in response to CNS injury or inflammation and it has been hypothesized that these factors may contribute to the resolution of tissue inflammation (Hohlfeld et al., 2006; Boche et al., 2006). TGF-β expression is increased during the recovery phase of EAE (Issazadeh et al., 1998) and TGF-β-treated microglia induce oligodendrocyte precursor cell migration (Lalive et al., 2005). Along with TGF-β, NGF production is increased in a variety of CNS disorders, including brain injury (Kossmann et al., 1996; Brown et al., 2004; DeKosky et al., 2004), EAE (De Simone et al., 1996), and multiple sclerosis relapsing phase (Laudiero et al., 1992). In addition, NGF, administered or delivered by transgenic T cells, delays clinical EAE onset in the marmoset and mice (Villoslada et al., 2000; Flugel et al., 2001). Since all these pathological conditions are associated with the presence of cytokines that stimulate the production of NGF by astrocytes, it has been hypothesized that NGF can act as an autocrine anti-inflammatory cytokine produced in the brain. These observations, along with the present results, lead us to hypothesize that the presence of NGF and TGF-β at sites of injury, where inflammatory cells as well as dying neurons are present (Lindholm et al., 1987; Crutcher et al., 1993), may promote microglial recruitment to the core of the lesion. Once recruited, microglial cells can be induced by these factors to acquire the anti-inflammatory and protective phenotype necessary to the efficient and silent removal of apoptotic bodies. In line with this hypothesis, we have shown that the specific interaction of microglial cells with apoptotic neurons attenuates the release of pro-inflammatory cytokines while promoting that of TGF-β and NGF (De Simone et al., 2003). It is possible that, through paracrine mechanisms, NGF and/or TGF-β may further recruit microglial cells to restore tissue homeostasis. Although other factors, including chemokines such as MCP-1, are abundantly released during inflammatory and immune-mediated diseases, and act as potent microglial chemoattractants (Ambrosini and Aloisi, 2004), the NGF/TGF-β induced migration of microglial cells may become relevant in other settings, characterized by the presence of neuronal apoptotic death such as during brain development or chronic diseases. 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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