Charles-Félix Calvo

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Sphingosine‐1‐phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg‐1 and Edg‐3. S1P stimulated thymidine incorporation and induced activation of extracellular signal‐regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P‐evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3‐kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin‐sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood–brain barrier.

Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by β-amyloid

Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)‐astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin‐1? (IL‐1?) and tumor necrosis factor‐? (TNF‐?) were identified as being the main factors responsible for this conditioned medium‐mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimers disease, at sites of β‐amyloid (A?) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL‐1? and TNF‐? activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

Inhibition of cytokine-induced connexin43 hemichannel activity in astrocytes is neuroprotective

Astrocytes express high levels of connexin43, a protein that forms two types of channels: gap junction channels for direct intercellular communication, and hemichannels for exchanges with the extracellular space. Inflammation induces connexin43 hemichannel activation, which has been proposed to be involved in neuroglial interactions. Here, we investigated the contribution of connexin43 to NMDA-induced excitotoxicity in neuron/astrocyte co-cultures, after treatment with a pro-inflammatory cytokine mixture, containing TNF-alpha and IL1-beta (Mix), that stimulated astroglial connexin43 hemichannel activity. Interestingly, NMDA treatment induced a higher amount of neurotoxicity in Mix-treated co-cultures than in untreated ones, whereas this extent of neurotoxicity was absent in enriched neuron cultures or in co-cultures with connexin43 knock-out astrocytes. Furthermore, application of connexin43 hemichannel blockers or a synthetic cannabinoid prevented the Mix-induced potentiated NMDA neurotoxicity. Altogether, these data demonstrate that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and suggest a neuroprotective role of connexin43 hemichannel blockade.

Brain macrophages inhibit gap junctional communication and downregulate connexin 43 expression in cultured astrocytes

Astrocytes are typically interconnected by gap junction channels that allow, in vitro as well as in vivo, a high degree of intercellular communication between these glial cells. Using cocultures of astrocytes and neurons, we have demonstrated that gap junctional communication (GJC) and connexin 43 (Cx43) expression, the major junctional protein in astrocytes, are controlled by neuronal activity. Moreover, neuronal death downregulates these two parameters. Because in several brain pathologies neuronal loss is associated with an increase in brain macrophage (BM) density, we have now investigated whether coculture with BM affects astrocyte gap junctions. We report here that addition of BM for 24 h decreases the expression of GJC and Cx43 in astrocytes in a density‐dependent manner. In contrast, Cx43 is not detected in BM and no heterotypic coupling is observed between the two cell types. A soluble factor does not seem to be involved in these inhibitions because they are not observed either in the presence of BM conditioned media or in the absence of direct contact between the two cell types by using inserts. These observations could have pathophysiological relevance as neuronal death, microglial proliferation and astrocytic reactions occur in brain injuries and pathologies. Because astrocyte interactions with BM and dying neurons both result in the downregulation of Cx43 expression and in the inhibition of GJC, a critical consequence on astrocytic phenotype in those situations could be the inhibition of gap junctions.

Hydrogen peroxide increases gap junctional communication and induces astrocyte toxicity: regulation by brain macrophages.

Cultured astrocytes are highly coupled by gap junction channels mainly constituted by connexin 43. We have previously shown that gap junctional communication (GJC) represents a functional property of astrocytes that is a target for their interaction with other brain cell types, including neurons and brain macrophages. In pathological situations, neurons as well as brain macrophages produce superoxide ions leading to the formation of hydrogen peroxide (H2O2) that can be cytotoxic. We report here that 10‐min exposure to 100 μM H2O2 increases GJC in astrocytes. Moreover, 30‐min exposure to 100 μM H2O2 induces, 24 h later, an astrocyte cell death by both apoptosis and necrosis. This H2O2‐induced astrocyte cell death is not affected when gap junctions are inhibited by several uncoupling agents, including 18α‐glycyrrhetinic acid, halothane, heptanol, and endothelin‐1, indicating that the proportion of cell death is not related to the level of GJC. The effect of H2O2 on gap junction channels does not result from the production of free radicals but is rather linked to modification of the redox equilibrium in astrocytes. Indeed, an oxidative agent reproduces the H2O2‐evoked response while reducing agents prevent the effect of H2O2. Finally, when astrocytes are cocultured with brain macrophages, the effects of H2O2 on both GJC and toxicity are not observed, revealing a new protective role of brain macrophages during oxidative stress.

Identification of an opioid peptide secreted by rat embryonic mixed brain cells as a promoter of macrophage migration

Conditioned media from embryonic mixed cells from the rat brain were used in a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17‐CM). Based upon checkerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow‐derived macrophages. The implication of a peptide compound in this activity was suggested by its resistance to heat as well as acid treatments, and by its sensitivity to aminopeptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI‐174 reduced the migration of BM in response to E17‐CM by 60%. This migratory activity was no longer effective when pertussis toxin‐treated BM were used. When the chemotactic effects of selective opioid agonists were compared to that of E17‐CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis indicated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met‐enkephalin‐like reactivity was found by RIA in the E17‐CM. Altogether, these observations suggest that a delta‐like opioid peptide released from embryonic mixed brain cells could be responsible for the infiltration of the developing CNS by macrophages precursors.

Identification of CSF‐1 as a brain macrophage migratory activity produced by astrocytes

Intraparenchymal migration of macrophages occurs in the CNS during development or as a consequence of tissue injuries. In the present study, we have shown, by using an in vitro chemotaxis assay, that cultured rat astrocytes obtained from the developing cerebral cortex and striatum produce soluble factors, which attract purified brain macrophages. The effect of astrocyte‐derived factors on macrophages was strongly reduced in the presence of antibodies neutralizing colony‐stimulating factor 1 (CSF‐1, also called M‐CSF), and recombinant CSF‐1 was found to act as a chemotactic agent on brain macrophages. Synthesis of CSF‐1 by cultured astrocytes was confirmed by northern detection of CSF‐1 transcripts. In contrast, the CSF‐1 gene was not expressed by cultured neurons from the cerebral cortex and striatum or by the brain macrophage population responsive to CSF‐1 gradient. ELISA detection of CSF‐1 in tissue extracts revealed the occurrence of this cytokine in the rat cerebral cortex during postnatal development and in adults. Altogether, our results demonstrate that astrocytes, through CSF‐1 secretion, can trigger the polarized migration of brain macrophages and suggest a new mechanism which could regulate the locomotion of these cells in the cerebral cortex during ontogenesis or following lesions. GLIA 24:180–186, 1998.

Albumin stimulates monocyte chemotactic protein-1 expression in rat embryonic mixed brain cells

Albumin, a blood protein absent from the adult brain in physiological situations, can be brought into contact with brain cells during development or, in adult, following breakdown of the blood–brain barrier occurring as a result of local inflammation. In the present study, we show that ovalbumin and albumin induce the release of monocyte chemotactic protein 1 (MCP‐1/CCL2) from rat embryonic mixed brain cells. A short‐term exposure to ovalbumin during the cell dissociation procedure is sufficient to generate MCP‐1 mRNA. A comparable effect is observed when the cells are incubated for 4 hr with ovalbumin or rat albumin, while MCP‐1 messengers are barely detectable following bovine albumin exposure. The amount of MCP‐1 protein measured in 4 hr‐supernatants of albumin‐treated cells followed the same albumin‐inducing pattern as that of MCP‐1 mRNA, while all albumins tested induced MCP‐1 protein after a 17 hr‐incubation period. The albumin‐induced MCP‐1 production is significantly inhibited in calphostin C‐treated cells, suggesting the implication of a protein kinase C‐dependent signaling pathway. This MCP‐1‐inducing activity is maintained after a lipid extraction procedure but abolished by proteinase K or trypsin treatments of albumin. The MCP‐1 secretion following albumin contact with nervous cells could thus interfere, by chemotactic gradient formation, with the brain infiltration program of blood‐derived cells during development or brain injury.

A pro- and an anti-inflammatory cytokine are synthetised in distinct brain macrophage cells during innate activation

Brain macrophages are known to exert dual and opposing functions on neuronal survival, which can be either beneficial or detrimental. The rationale of our study is that this duality could arise from an exclusive secretion of either pro- or anti-inflammatory cytokine by distinct cell subsets, cytokines that could respectively mediate neurotoxic or neurotrophic effects. Innate immune response was induced in macrophage cultures prepared from embryonic-day-16 to postnatal-day-8 mouse brains. By immunofluorescent detection of intracellular cytokines, we have assessed the occurrence of TNFalpha or IL10 synthesis at single cell level and observed distinct secretory patterns that include cells producing exclusively TNFalpha or IL10, cells producing both cytokines and non-producer cells. These secretory patterns are differentially regulated by MAP-kinase inhibitors. Altogether, these results demonstrate that synthesis of either a pro- or an anti-inflammatory cytokine can segregate distinct brain macrophages and suggests a functional cell-subset-specialisation.

Neurons and brain macrophages regulate connexin expression in cultured astrocytes.

Neurons and brain macrophages (BM), respectively, increase and inhibit gap junctional communication (GJC) and connexin expression in cultured astrocytes. Thus, in brain diseases and injuries, neuronal death associated with the BM activation may decrease GJC in astrocytes and therefore have a physiopathological relevance.