Elena Ambrosini

Chemokines and glial cells: a complex network in the central nervous system.

Chemokines are small secreted proteins that are essential for the recruitment and activation of specific leukocyte subsets at sites of inflammation and for the development and homeostasis of lymphoid and nonlymphoid tissues. During the past decade, chemokines and their receptors have also emerged as key signaling molecules in neuroinflammatory processes and in the development and functioning of the central nervous system. Neurons and glial cells, including astrocytes, oligodendrocytes, and microglia, have been identified as cellular sources and/or targets of chemokines produced in the central nervous system in physiological and pathological conditions. In this article, we provide an update of chemokines and chemokine receptors expressed by glial cells focusing on their biological functions and implications in neurological diseases.

Lymphoid Chemokines CCL19 and CCL21 are Expressed in the Central Nervous System During Experimental Autoimmune Encephalomyelitis: Implications for the Maintenance of Chronic Neuroinflammation

The simultaneous presence of dendritic, T‐ and B‐cells in the central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, suggests that interactions among these cell types might be instrumental in the local induction and maintenance of autoimmune reactions. In this study, we explored the possibility that such aberrant leukocyte recruitment in the CNS could be sustained by “lymphoid” chemokines which orchestrate dendritic cell and lymphocyte homing to lymphoid organs. Transcripts for CCL19 and CCL21 and their common receptor CCR7 were induced in the CNS of mice undergoing relapsing‐remitting and chronic‐relapsing EAE. While CCL21 immunoreactivity was confined to the endothelium of some inflamed blood vessels, CCL19 was expressed by many infiltrating leukocytes and some astrocytes and microglia in the CNS parenchyma. CCR7+ cells accumulated in inflammatory lesions during EAE progression, when abundant infiltration of the CNS by mature dendritic cells, B‐cells and cells expressing naive T‐cell markers also occurred. These findings suggest that CCL19 and CCL21 produced in the EAE‐affected CNS may be critical for the homing of antigen presenting cells and lymphocytes, resulting in continuous local antigenic stimulation and maintenance of chronic neuroinflammation.

Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions.

As a result of their close association with the blood-brain barrier, astrocytes play an important role in regulating the homing of different leukocyte subsets to the inflamed central nervous system (CNS). In this study, we investigated whether human astrocytes produce chemokines that promote the migration of myeloid dendritic cells (DCs). By reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, we show that cultured human astrocytes stimulated with interleukin-1β and tumor necrosis factor produce CCL2, CCL3, CCL4, CCL5, CCL20, and CXCL12 that act on immature DCs, but not CCL19 and CCL21, 2 chemokines specific for mature DCs. Compared with controls, supernatants of cytokine-stimulated astrocytes are more effective in promoting the migration of immature monocyte-derived DCs (iMDDCs). Desensitization of CXCR4 (receptor for CXCL12), CCR1-3-5 (shared receptors for CCL3-4-5), and CCR6 (receptor for CCL20) on iMDDC reduces cell migration toward astrocyte supernatants, indicating that astrocytes release biologically relevant amounts of iMDDC-attracting chemokines. By immunohistochemistry, we show that CXCL12 and, to a lesser extent, CCL20 are expressed by reactive astrocytes in multiple sclerosis lesions. These data lend support to the idea that astrocyte-derived chemokines may contribute to immature DC recruitment to the inflamed CNS.

Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development

To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X1,2,3,4,7) and P2Y (P2Y1,2,4) receptors. Intracellular Ca2+ recording by Fura‐2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPβS = ADP = Benzoyl ATP > ATP > ATPγS > UTP, α,β‐meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of α,βmeATP‐induced inward current in whole‐cell recording, P2X7 and P2Y1 were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y1‐specific agonist ADPβS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet‐derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y1 antagonist MRS2179. By confocal laser scanning microscopy, P2Y1 receptors were localized in NG2‐labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y1 receptors.

Astrocytes are the major intracerebral source of Macrophage inflammatory protein-3α/CCL20 in relapsing experimental autoimmune encephalomyelitis and in vitro

Macrophage inflammatory protein‐3α/CCL20 is a recently identified chemokine that binds to CCR6 and acts as a chemoattractant for memory/differentiated T‐cells, B‐cells, and immature dendritic cells. We have previously reported that CCL20 and CCR6 mRNAs are expressed in the CNS of SJL mice with experimental autoimmune encephalomyelitis (EAE) and that CCL20 is produced by CNS‐infiltrating leukocytes at disease onset and, additionally, by intraparenchymal astrocyte‐like cells during disease relapses. In this study, we provide further immunohistochemical evidence that astrocytes represent the main CNS source of CCL20 during EAE. Moreover, we show that the proinflammatory cytokines interleukin‐1β and tumor necrosis factor‐α, but not interferon‐γ, induce expression of CCL20 mRNA and secretion of CCL20 protein in cultures of mouse brain‐derived astrocytes. We also show that supernatants from cytokine‐activated astrocytes stimulate the migration of polarized T helper cells and that this effect is partially inhibited by anti‐CCL20 antibody. These findings suggest that, through secretion of CCL20, astrocytes could play an important role in orchestrating the recruitment of specific leukocyte subsets to the inflamed CNS and in regulating CNS‐targeted immune responses. GLIA 41:290–300, 2003.

Functional characterization of substance P receptors on cultured human spinal cord astrocytes: synergism of substance P with cytokines in inducing interleukin-6 and prostaglandin E2 production.

Following brain injury, astrocytes express receptors for cytokines and neuropeptides and secrete several regulatory mediators that have a well established role in inflammation, immunity, and tissue development or repair. To elucidate the role of substance P (SP), a neurotransmitter peptide of the tachykinin family, in inducing astrocyte secretory activities, we have examined the expression of SP receptors and the functional consequences of their activation in cultured astrocytes from the human embryonic brain or spinal cord. Radioligand binding studies revealed that only one type of SP receptors, the high affinity NK‐1 receptor, was present on human astrocytes and that spinal cord astrocytes expressed about 6 times as many SP binding sites as brain astrocytes. Following SP treatment, a substantial inositol phosphate formation was observed in spinal cord astrocytes only. Stimulation of spinal cord astrocytes with SP alone did not induce secretion of cytokines [interleukin‐6 (IL‐6), granulocyte‐macrophage‐CSF, macrophage chemoattractant protein‐1 or leukemia inhibitory factor] or prostaglandin E2 (PGE2). Interestingly, however, SP selectively potentiated the inducing effect of IL‐1β on IL‐6 and PGE2 secretion by spinal cord astrocytes without affecting the IL‐1‐β‐evoked secretion of other cytokines. SP also enhanced the small inducing effect of tumor necrosis factor‐α (TNF‐α) on IL‐6 and PGE2 secretion and that of transforming growth factor‐β on PGE2 secretion. These results suggest that SP can enhance immunoregulatory and neurotrophic astroglial functions mediated by IL‐6 and PGE2 by acting in concert with a set of cytokines whose cerebral expression has been reported during development and in a variety of diseases. GLIA 21:183–193, 1997.

Induction of macrophage-derived chemokine/CCL22 expression in experimental autoimmune encephalomyelitis and cultured microglia: implications for disease regulation

Macrophage-derived chemokine (MDC/CCL22) and its receptor CCR4 have been implicated in chronic inflammatory processes and in the homing of monocytes, Th2 cells and regulatory T-cell subsets. Here, we demonstrate that MDC and CCR4 mRNAs are expressed in the central nervous system (CNS) of mice developing relapsing-remitting and chronic-relapsing forms of experimental autoimmune encephalomyelitis (EAE). By immunohistochemistry, we show that MDC is produced by CNS-infiltrating leukocytes and intraparenchymal microglia, whereas CCR4 is expressed on some invading leukocytes. Upon in vitro activation, mouse microglia express MDC transcripts and secrete bioactive MDC that induces chemotaxis of Th2, but not Th1 cells. We suggest that MDC produced by microglia could regulate Th1-mediated CNS inflammation by facilitating the homing of Th2 and, possibly, regulatory T cells into the lesion site.

Lymphoid chemokines in chronic neuroinflammation

Abstract Lymphoid chemokines play an essential role in the establishment and maintenance of lymphoid tissue microarchitecture and have been implicated in the formation of tertiary (or ectopic) lymphoid tissue in chronic inflammatory conditions. Here, we review recent advances in lymphoid chemokine research in central nervous system inflammation, focusing on multiple sclerosis and the animal model experimental autoimmune encephalomyelitis. We also highlight how the study of lymphoid chemokines, particularly CXCL13, has led to the identification of intrameningeal B-cell follicles in the multiple sclerosis brain paving the way to the discovery that these abnormal structures are highly enriched in Epstein–Barr virus-infected B cells and plasma cells.

NGF promotes microglial migration through the activation of its high affinity receptor: Modulation by TGF-β

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 (< or =0.2 ng/ml) of transforming growth factor-beta (TGF-beta), 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-beta (> or =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-beta. Our observations suggest that both NGF and TGF-beta 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.

The β1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis

Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.