Michel Mallat

Cytotoxic Effect of Brain Macrophages on Developing Neurons

Brain macrophages are transiently present in different regions of the central nervous system during development or in the course of tissue remodelling following various types of injuries. To investigate the influence of these phagocytes on neuronal growth and survival, brain macrophages stemming from the cerebral cortex of rat embryos were added to neuronal primary cultures. A neurotoxic effect of brain macrophages was demonstrated by the reduction of the number of neurons bearing neurites within two days of contact between the two cell types. Neuronal death and phagocytosis were also directly observed in video recordings of living cultures. This toxicity involved the production by brain macrophages of reactive oxygen intermediates, as shown by the protective effect of catalase, a scavenger of H2O2. In addition, the respiratory bursts of brain macrophages were stimulated in the presence of neurons. These results suggest that brain macrophages could favour the appearance of neuroregressive events which occur either during neurogenesis or in neurodegenerative diseases, implying intracerebral recruitment of mononuclear phagocytes.

Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase

Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O2·−). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22phox, NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22phox] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O2·− in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O2·−, which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1β. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1- and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.

System Xc− and Apolipoprotein E Expressed by Microglia Have Opposite Effects on the Neurotoxicity of Amyloid-β Peptide 1–40

Because senile plaques in Alzheimers disease (AD) contain reactive microglia in addition to potentially neurotoxic aggregates of amyloid-β (Aβ), we examined the influence of microglia on the viability of rodent neurons in culture exposed to aggregated Aβ 1–40. Microglia enhanced the toxicity of Aβ by releasing glutamate through the cystine-glutamate antiporter system Xc−. This may be relevant to Aβ toxicity in AD, because the system Xc−-specific xCT gene is expressed not only in cultured microglia but also in reactive microglia within or surrounding amyloid plaques in transgenic mice expressing mutant human amyloid precursor protein or in wild-type mice injected with Aβ. Inhibition of NMDA receptors or system Xc− prevented the microglia-enhanced neurotoxicity of Aβ but also unmasked a neuroprotective effect of microglia mediated by microglial secretion of apolipoprotein E (apoE) in the culture medium. Immunodepletion of apoE or targeted inactivation of the apoE gene in microglia abrogated neuroprotection by microglial conditioned medium, whereas supplementation by human apoE isoforms restored protection, which was potentiated by the presence of microglia-derived cofactors. These results suggest that inhibition of microglial system Xc− might be of therapeutic value in the treatment of AD. Its inhibition not only prevents glutamate excitotoxicity but also facilitates neuroprotection by apoE.

Production of Monocyte Chemotactic Protein‐1 By Rat Brain Macrophages

In the present study, we show that cultured rat brain macrophages release a soluble factor that stimulates the migration of bone marrow‐derived macrophages, as determined by an in vitro chemotaxis assay. A checkerboard analysis indicated that most of this effect resulted from a polarized migration of the cells (chemotactic phenomenon), rather than in an increase in cell motility (chemokinesis). This activity was significantly decreased by an immune serum directed against the rat monocyte chemoattractant protein‐1 (chernokine MCP‐1). Northern blot analysis demonstrated expression of the MCP‐1 gene in cultured brain macrophages, but its absence in unstimulated bone marrow‐derived macrophages. Up‐regulation of MCP‐1 expression was observed when lipopolysaccharide was added to cultured brain macrophages, a peak occurring after a 6 h period of stimulation. Also, inflammatory cytokines such as interleukin (IL)‐1β, colony stimulating factor‐1, tumour necrosis factor‐α and IL‐6 individually increased the basal level of MCP‐1 mRNA. Subsequently, we demonstrated the in vivo production of MCP‐1 in the adult rat brain following injury induced by a local injection of kainic acid. MCP‐1 synthesis was localized in both astrocytes and brain macrophages. These results suggest that the activation of resting microglial cells into brain macrophages and their subsequent secretion of chemokines could contribute to the mechanism(s), leading to the infiltration of the CNS by blood‐derived monocytes, as observed in several pathologies.

Interleukin 1 and Tumor Necrosis Factor-α Stimulate the Production of Colony-Stimulating Factor 1 by Murine Astrocytes

Abstract: Astrocytes have the ability to secrete colony‐stimulating factor 1 (CSF‐1), a growth factor known to stimulate the proliferation of brain macrophages. We have studied the effect of cytokines such as interleukin 1 (IL‐1), tumor necrosis factor‐α (TNFα), and interleukin 6 (IL‐6) on the production of CSF‐1 by cultured primary astrocytes and an astrocytic cell line derived from embryonic mouse brain. We observed that both TNFα and IL‐1 increased CSF‐1 mRNA and protein levels in the astrocytic cultures. In contrast, IL‐6 was ineffective. The CSF‐1 mRNA levels were strongly reduced by incubating immortalized astrocytic cells with staurosporine, a protein kinase C inhibitor, both in the absence and in the presence of cytokines. Conversely, 12‐O‐tetradecanoylphorbol 13‐acetate, a protein kinase C activator, increased CSF‐1 mRNA levels. These results suggest a mechanism whereby mononuclear phagocytes could favor their own recruitment in the CNS by producing cytokines.

Immunohistochemical detection of thrombospondin in microglia in the developing rat brain

The development of microglia involves the expression of a phenotype displaying phagocytic behaviour termed brain macrophage or amoeboid microglial cell. We have previously shown that rat brain macrophages purified in vitro secrete thrombospondin, an extracellular matrix protein, which acts on cultured neuronal cells by promoting neurite growth. In the present study, the expression of thrombospondin was investigated in tissue sections of the developing rat forebrain in relation to the distribution of microglia. These cells were identified using anti-macrophage antibodies and the isolectin B4 from Bandeiraea simplicifolia. Immunocytochemical detection of thrombospondin clearly outlined a cell population displaying the morphologies and distribution of brain macrophages, from the 17th day of embryonic life up to the end of the second postnatal week. These cells were most numerous in cortical and subcortical regions of developing fibre tracts such as the corpus callosum or the internal capsule. The localization of thrombospondin in brain macrophages was confirmed by double immunostaining using ED1 monoclonal anti-macrophage antibodies. Ramified microglial cells were also labelled transiently by anti-thrombospondin antibodies during early postnatal life. These results provide in situ evidence supporting the notion that microglial cells could favour axonal growth by producing thrombospondin during development.

Survival of intracerebrally grafted rat dopamine neurons previously cultured in vitro

Fetal rat dopamine (DA) neurons were cultured in vitro for a 6-day period and transported, after redissociation, for 2 days prior to being grafted to the neostriatum of adult rats with 6-hydroxydopamine lesions of the ascending nigrostriatal pathway. In 2 of the 5 graft recipients that were tested for amphetamine-induced motor asymmetry, the grafts eliminated the lesion-induced turning behaviour within 3-6 weeks after transplantation. Fluorescence histochemistry revealed surviving grafts in all 6 recipients at 7 weeks after transplantation, containing between 42 and 125 DA neurons. The number of surviving DA neurons in the 3 non-compensated rats was below the minimum number of cells previously found to be necessary for functional effects on turning behaviour to occur.

[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [3H]metergoline characterized by a KD of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7‐dihydroxy‐tryptamine‐ and kainic acid‐induced lesions indicated that this specific binding site was localized post‐synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [3H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad‐renaline for displacing bound [3H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [3H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [3H]metergoline and [3H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7‐dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [3H]metergoline might well be the appropriate ligand for studying the characteristics of the ‘antagonist form’ of serotonin receptors postulated by Bennett and Snyder.

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. I. Modulation of Central 5-Hydroxytryptamine Receptors in the Rat

Abstract: The present study indicates that central 5‐hydroxytryptamine (5‐HT; serotonin) receptors can be modulated in opposite directions by Ca2+ and guanine nucleotides [guanosine triphosphate (GTP), β, γ‐imidoguanosine 5′‐triphosphate (GppNHp)]. Thus CaCl2 (≥0.5 mm) inhibited whereas GTP and GppNHp (10 μm) stimulated the 5‐HT‐sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (Kd−1) and the number (Bmax) of [3H]5‐HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca2+ (≥0.25 mm); GTP (≥0.1 mm) and GppNHp (≥0.3; μm) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [3H]5‐HT binding was counteracted by Ca2+: the addition of this cation (5mm‐CaCl2) to the assay mixture resulted in a 40‐fold increase in the IC50 for GTP; the IC50 for GppNHp increased five‐fold under the same condition. The examination of the respective effects of Ca2+ and of GTP on the specific binding of [3H]5‐HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca2+‐induced increase and of the GTP‐induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid‐treated rats because [3H]5‐HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca2+‐induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[3H]5‐HT binding sites to adenylate cyclase in the rat brain.

Influence of interleukin-1 and tumor necrosis factor alpha on the growth of microglial cells in primary cultures of mouse cerebral cortex: involvement of colony-stimulating factor 1

The influence of monokines and CNS-derived colony-stimulating factors (CSF) on the growth of microglia has been studied in mixed glial primary cultures stemming from mouse embryos. We observed that spontaneous growth of microglial cells in the presence of astrocytes is blocked by adding anti-colony-stimulating factor 1 (CSF-1) antibodies to the cultures. Both interleukin-1 (IL-1) and tumor necrosis factor-alpha(TNF alpha) strongly increased the number of microglial cells in mixed glial cultures and this effect was prevented by anti-CSF-1 antibodies. In contrast, anti-interleukin-3 (IL-3) or anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies did not significantly affect the in vitro growth of microglia. These results provide functional significance to astrocytic productions of CSF-1 and their modulations by IL-1 or TNF alpha.