Cristina Agresti

Reversible Inhibitory Effects of Interferon‐γ and Tumour Necrosis Factor‐α on Oligodendroglial Lineage Cell Proliferation and Differentiation In Vitro

We have investigated the effects of the two prominent inflammatory cytokines, interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum‐free medium and exposed to IFN‐γ (1‐100 U/ml), TNF‐α (25‐100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day 3 or from day 3 to day 6. While cell survival was not affected in any of the conditions tested, IFN‐γ dose‐dependently inhibited [3H]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT; by up to 33%). TNF‐α synergized with IFN‐γ, but was ineffective by itself. Moreover, IFN‐γ totally antagonized the induction by basic fibroblast growth factor and platelet‐derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN‐γ also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN‐γ was potentiated by TNF‐α, which was ineffective when tested alone. The inhibitory activity of IFN‐γ was rapidly reversible: 3 days after removal of the cytokine, administered from day 1 to day 3, complete recovery of cell proliferation and differentiation could be documented. The cytokine‐induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription‐polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days 3 to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN‐γ, and further depressed in cultures treated with IFN‐γ and TNF‐α, while TNF‐α alone was ineffective. The above observations may help explain the role of IFN‐γ and TNF‐α in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing‐remitting course of the disease.

Nuclear Factor kB-independent Cytoprotective Pathways Originating at Tumor Necrosis Factor Receptor-associated Factor 2

Most normal and neoplastic cell types are resistant to tumor necrosis factor (TNF) cytotoxicity unless cotreated with protein or RNA synthesis inhibitors, such as cycloheximide and actinomycin D. Cellular resistance to TNF requires TNF receptor-associated factor 2 (TRAF2), which has been hypothesized to act mainly by mediating activation of the transcription factors nuclear factor kB (NFkB) and activator protein 1 (AP1). NFkB was proposed to switch on transcription of yet unidentified anti-apoptotic genes. To test the possible existence of NFkB-independent cytoprotective pathways, we systematically compared selective trans-dominant inhibitors of the NFkB pathway with inhibitors of TRAF2 signaling for their effect on TNF cytotoxicity. Blockade of TRAF2 function(s) by signaling-deficient oligomerization partners or by molecules affecting TRAF2 recruitment to the TNF receptor 1 complex completely abrogated the cytoprotective response. Conversely, sensitization to TNF cytotoxicity induced by a selective NFkB blockade affected only a fraction of TNF-treated cells in an apparently stochastic manner. No cytoprotective role for c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), which are activated by TRAF2 and contribute to stimulation of activator protein 1 activity, could be demonstrated in the cellular systems tested. Although required for cytoprotection, TRAF2 is not sufficient to protect cells from TNF + cycloheximide cytotoxicity when overexpressed in transfected cells, thus indicating an essential role of additional TNF receptor 1 complex components in the cytoprotective response. Our results indicate that TNF-induced cytoprotection is a complex function requiring the integration of multiple signal transduction pathways.

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.

Synergistic stimulation of MHC class I and IRF-1 gene expression by IFN-gamma and TNF-alpha in oligodendrocytes

In order to understand the molecular basis of the synergistic action of interferon γ (IFN‐γ) and tumour necrosis factor α (TNF‐α) on rat oligodendrocyte development, we studied some aspects of the signalling pathways involved in the regulation of the major histocompatibility complex (MHC) class I and the interferon regulatory factor 1 (IRF‐1) gene expression. Two well‐defined inducible enhancers of the MHC class I gene promoter, the MHC class I regulatory element (MHC‐CRE) and the interferon consensus sequence (ICS), were analysed. Neither IFN‐γ nor TNF‐α was capable of inducing MHC‐CRE binding activity when administrated alone. Following the exposure of oligodendrocytes to IFN‐γ, TNF‐R1 expression was transcriptionally induced by the binding of signal transducer and activator of transcription (STAT‐1) homodimers to the IFN‐γ activated site (GAS) present in the gene promoter. The upregulation of TNF‐R1 allowed TNF‐α to induce the binding of nuclear factor‐κB (NF‐κB) to the MHC‐CRE site. With respect to ICS element, IFN‐γ induced IRF‐1 binding, that was further enhanced upon co‐treatment with TNF‐α. The existence of a synergism between IFN‐γ and TNF‐α in stimulating IRF‐1 expression at the transcriptional level was supported by IRF‐1 promoter analysis: IFN‐γ directly induced the binding of STAT‐1 homodimers to the GAS element, while NF‐κB binding to the κB sequence was activated by TNF‐α only after IFN‐γ treatment. This transcriptional regulation of IRF‐1 gene by IFN‐γ and TNF‐α was confirmed at the mRNA level. The synergism demonstrated in the present study highlights the importance of cytokine interactions in magnifying their biological effects during brain injury and inflammation.

Activation of TNF receptor 2 in microglia promotes induction of anti-inflammatory pathways

Fine regulation of the innate immune response following brain injury or infection is important to avoid excessive activation of microglia and its detrimental consequences on neural cell viability and function. To get insights on the molecular networks regulating microglia activation, we analyzed expression, regulation and functional relevance of tumor necrosis factor receptors (TNFR) 2 in cultured mouse microglia. We found that microglia upregulate TNFR2 mRNA and protein and shed large amounts of soluble TNFR2, but not TNFR1, in response to pro-inflammatory stimuli and through activation of TNFR2 itself. By microarray analysis, we demonstrate that TNFR2 stimulation in microglia regulates expression of genes involved in immune processes, including molecules with anti-inflammatory and neuroprotective function like granulocyte colony-stimulating factor, adrenomedullin and IL-10. In addition, we identify IFN-γ as a regulator of the balance between pro- and anti-inflammatory/neuroprotective factors induced by TNFR2 stimulation. These data indicate that, through TNFR2, microglia may contribute to the counter-regulatory response activated in neuropathological conditions.

Heterotypic and homotypic cellular interactions influencing the growth and differentiation of bipotential oligodendrocyte-type-2 astrocyte progenitors in culture☆

Cell populations highly enriched in oligodendrocyte-type-2 astrocyte (O-2A) progenitors (so defined by their ability to bind the monoclonal antibodies LB1 and O4, and by the lack of expression of the differentiated glial markers galactocerebroside and glial fibrillary acidic protein (GFAP) were obtained from rat mixed cortical glial cultures. The O-2A progenitors were grown at low density (2 X 10(4) cells/cm2) in BME + 10% fetal calf serum (FCS) on a poly-L-lysine (PLL) substrate (controls) or on a substrate of purified type-1 astrocytes (AS) killed by air drying (K-AS), in order to analyze the effects of the interaction between the two cell types on the growth and differentiation of the immature O-2A cells, independently of the mitogenic soluble factors (e.g., platelet-derived growth factor; see Raff, 1989, Science 243, 1450-1455) secreted by type-1 AS. While on PLL most of the progenitors differentiated into GFAP+ type-2 AS within 1 week, on K-AS they largely differentiated into GalC+ oligodendrocytes (OL). On the latter substrate, however, the precursors achieved a higher density, due to higher proliferative activity. The additional observation, that when immature O-2A cells were seeded at high density (greater than 5 X 10(4) cells/cm2) on PLL their differentiation into OL was much more pronounced than in cultures of lower density, indicates that there is a close correlation between the density of immature O-2A cells and lineage decision, and that the increased OL differentiation of the immature O-2A cells on K-AS is at least partly related to the higher density achieved by the cells on this substrate. The enhanced proliferation of immature O-2A cells on K-AS did not appear to be related to platelet-derived growth factor or fibroblast growth factor remaining attached to the substrate, nor to known components of the extracellular matrix (ECM), such as heparan sulfate, chondroitin sulfate, laminin, or fibronectin, but was probably due to other components of a polypeptide nature present in the ECM produced by type-1 AS. A cell-free ECM was in fact almost as mitogenic as the K-AS substrate, and the mitogenic activities of both K-AS and AS-ECM were similarly inhibited by a set of enzymatic (pronase, trypsin) and physicochemical (heat, pH) treatments.

TNF receptor 2 protects oligodendrocyte progenitor cells against oxidative stress.

The neuroprotective role of TNF receptor 2 (TNFR2) has been shown in various studies. However, a direct role of TNFR2 in oligodendrocyte function has not yet been demonstrated. Using primary oligodendrocytes of transgenic mice expressing human TNFR2, we show here that TNFR2 is primarily expressed on oligodendrocyte progenitor cells. Interestingly, preconditioning with a TNFR2 agonist protects these cells from oxidative stress, presumably by increasing the gene expression of distinct anti-apoptotic and detoxifying proteins, thereby providing a potential mechanism for the neuroprotective role of TNFR2 in oligodendrocyte progenitor cells.

HIV-gp120 affects the functional activity of oligodendrocytes and their susceptibility to complement

The aim of this study was to assess whether the HIV protein gp120 can induce direct or/and indirect damage to oligodendrocytes (OL). Using highly purified cultures of rat OL, we report that gp120 binds to OL and induces functional alterations in these cells. Indeed, the percentage of cells expressing myelin basic protein (MBP) and the levels of all four MBP isoforms were substantially reduced after a 3‐day treatment with 10 nM gp120. As gp120 depressed the ability of OL to reduce the tetrazolium salt MTT (a sign of mitochondrial impairment), the alteration of MBP production may be a consequence of decreased metabolic activity. The above effects were accompanied by a small increase in the number of apoptotic nuclei (from 4.3% in controls to 17.6% in cells treated for 3 days with gp120). As complement can lyse OL and gp120 is known to activate complement, we also studied the interaction between these two factors using OL cultures. The viral protein potentiated (by about 25%) the lytic effect of complement, when administered to the cultures 5 hr after complement, and depressed it (by about 30–40%), when added 5 hr before complement. Heat denaturation and anti‐gp120 antibodies prevented the direct effect of gp120 on OL, but did not influence the interactions between gp120 and complement. Some gp120 non glycosylated peptides (V3 loop, 254‐274 and 415‐435 peptides) mimicked the ability of gp120 to antagonize the lytic effect of complement, but not that of potentiating complement lytic activity. In conclusion, our study indicates that gp120 can alter OL functional activity directly and can interfere with OL susceptibility to complement mediated lysis. J. Neurosci. Res. 50:946–957, 1997.

Is the oligodendroglial differentiation of bipotential oligodendrocyte-type 2 astrocyte progenitors promoted by autocrine factors?

When immature O-2A (oligodendrocyte-type 2 astrocyte) lineage cells purified from rat mixed cortical glial cultures were subcultured at low density in 10% fetal calf serum (FCS)-containing medium they largely differentiated into type-2 astrocytes. When the same number of cells was subcultured at high density in the same volume of medium the proportion of O-2A progenitors differentiating into oligodendrocytes was substantially increased. The possibility that oligodendrocyte differentiation in high-density cultures is facilitated by autocrine factors is supported by the observation that a medium conditioned by high-density subcultures of purified O-2A cells contains high molecular weight (greater than 30 kDa), non-mitogenic factor(s) capable of inducing a rapid differentiation of immature 0-2A cells into oligodendrocytes even in low-density cultures.

HIV-1 myristoylated nef treatment of murine microglial cells activates inducible nitric oxide synthase, NO2 production and neurotoxic activity

Background The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors. Methodology/Principal Findings We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells. Conclusions These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.