A Culture Model of Reactive Astrocytes: Increased Nerve Growth Factor Synthesis and Reexpression of Cytokine Responsiveness

Abstract: Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1β and interferon-γ, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

Synergistic Activation of DNA Synthesis in Astrocytes by Fibroblast Growth Factors and Extracellular ATP

Abstract: The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet-derived growth factor or epidermal growth factor. ATP appears to act via P₂ purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis-resistant P₂ agonists, ADPβS and ATPγS. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.     

Transduction Mechanisms Involved in Thrombin Receptor-Induced Nerve Growth Factor Secretion and Cell Division in Primary Cultures of Astrocytes

Abstract: In astrocytes, thrombin and thrombin receptor-activating activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [³H]thymidine incorporation into DNA (EC₅₀ = 1 n M and +650% at 100 n M for thrombin; EC₅₀ = 3 µ M and +600% at 100 µ M for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 n M thrombin or 100 µ M TRAP-14). The [³H]thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as G_i or G_o, is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

IFN-γ and IL-4 differentially shape metabolic responses and neuroprotective phenotype of astrocytes

Astrocytes can either exacerbate or ameliorate secondary degeneration at sites of injury in the CNS but the contextual basis for eliciting these opposing phenotypes is poorly understood. In this study, we demonstrate that the two major cytokines produced by Th1 and Th2 cells, interferon-γ (IFN-γ), and interleukin-4 (IL-4), respectively, contribute differentially to shaping a neuroprotective response in astrocytes. While IFN-γ protects the ability of oxidatively stressed murine astrocytes to clear extracellular glutamate in culture, IL-4 has no effect at any concentration that was tested (10–100 ng/mL). The enhanced release of neuroprotective thiols and lactate by astrocytes in response to T cell stimulation is mimicked by both IL-4 and IFN-γ. When co-administered, IL-4 abrogated the protective effect of low IFN-γ on the glutamate clearance function of oxidatively stressed astrocytes in a dose-dependent manner. Astrocyte-conditioned media obtained from cells cultured in the presence of IL-4 (10 or 100 ng/mL) or IFN-γ (10 ng/mL) decreased by ∼2-fold, neuronal apoptosis induced by oxidative stress in vitro . However, unlike IL-4, IFN-γ at high concentrations (100 ng/mL) was not neuroprotective. Our studies with IFN-γ and IL-4 suggest that a balanced Th1 and Th2 cytokine response might be needed for protecting two key astrocytic functions, glutamate clearance and thiol secretion and might be pertinent to neuroprotective approaches that are aimed at inhibition of an initial pro-inflammatory response to injury or its sustained boosting.     

Inhibitory effects of aripiprazole on interferon-gamma-induced microglial activation via intracellular Ca2+ regulation in vitro

The activation of the inflammatory/immunological response system is suggested to be related to the pathophysiology of schizophrenia. Aripiprazole is a novel atypical antipsychotic, which is a high-affinity dopamine D₂ receptor partial agonist. Atypical antipsychotics, all of which have dopamine D₂ receptor antagonism, have recently reported to have significantly inhibitory effects on interferon (IFN)-γ-induced microglial activation in vitro . In the present study, we investigated whether or not aripiprazole also has anti-inflammatory effect on IFN-γ-induced microglial activation. Not quinpirole, dopamine D₂ full agonist, but aripiprazole significantly inhibited the generation of nitric oxide (NO) and tumor necrosis factor (TNF)-α from IFN-γ-activated microglia and suppressed the IFN-γ-induced elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in murine microglial cells. Increased [Ca²⁺]_i has been reported to be required, but by itself not sufficient, for the release of NO and certain cytokines. As a result, we can speculate that aripiprazole may inhibit IFN-γ-induced microglial activation through the suppression of IFN-γ-induced elevation of [Ca²⁺]_i in microglia. Our results demonstrated that not only antipsychotics which have dopamine D₂ receptor antagonism but also aripiprazole have anti-inflammatory effects via the inhibition of microglial activation. Antipsychotics may therefore have a potentially useful therapeutic effect on patients with schizophrenia by reducing the microglial inflammatory reactions.     

Mechanisms of α-Sialosyl Cholesterol Action to Suppress Both Cyclic AMP Production and DNA Synthesis of Rat Glial Cells

Abstract: α-Sialosyl cholesterol (α-SC) that elicited morphological differentiation of rat astrocytes not only lowered intracellular cyclic AMP (cAMP) levels but also inhibited cAMP production induced by either α-isoproterenol, cholera toxin, or forskolin. The targets of α-SC in the cAMP production system of rat astrocytes were investigated to understand the mechanism of the a-SC action on cAMP production. cAMP production evoked by a-isoproterenol (1 μ M ) was entirely canceled by β blockers such as propranolol and timolol (1 μ M ), but not by α-SC. Concentrations of α-SC greater than 15 μ M were required for 50% inhibition of the activation by a β agonist. Although α-SC inhibited in a dose-dependent manner the activities of membrane-associated adenylate cyclase that had been stimulated by either GTPγS or forskolin, α-SC inhibited neither GTP-binding activities nor GTPase activities of the membrane-associated G proteins. These findings suggest that α-SC suppresses adenylate cyclase directly, but not β receptors or G proteins, and that it promotes the morphological differentiation of rat astrocytes through a mechanism regulating directly the cytoskeletal organization, regardless of intracellular cAMP level. α-SC (30 μ M ) suppressed 40% of DNA synthesis in the cell-free system, which contained the cytosolic extracts and the nucleus fraction prepared from rat astrocytoma C6 cells. Approximately 25% of α-SC incorporated in the astrocyte cytoplasm was transferred to the nuclei by 10 min after the addition. Thus, it is likely that α-SC that had been incorporated in the cytosol suppressed adenylate cyclase by acting from the cytosolic side of the plasma membrane, and separately suppressed nuclear DNA synthesis.     

Reliability of laboratory models in the analysis of TBP2 and other meningococcal antigens

Abstract Lipoarabinomannan derived from the virulent Erdman strain and a rapidly growing, laboratory-attenuated strain of Mycobacterium tuberculosis were evaluated for their ability to modulate the production of nitric oxide (NO) by macrophages activated with IFN-γ or IFN-γ and LPS. It was observed that in macrophages pretreated with 100 μg ml⁻¹ LAM, the NO induced by IFN-γ alone was augmented while the NO induced by IFN-γ and LPS was reduced. LAM was also shown to synergize with IFN-γ in the induction of NO, with AraLAM from the attenuated strain exhibiting greater potency than ManLAM from the Erdman strain. Despite the modulation of NO production, LAM did not affect the IFN-γ-induced macrophage growth inhibition of Francisella tularensis LVS, an organism whose growth inhibition in activated macrophages is dependent upon NO.     

Regulation by Interleukin-1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.     

Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor-Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC-γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC-γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC-γ1 (PLC-γ1PC12), the SH2-SH2-SH3 domain (PLC-γ1SH223PC12), SH2-SH2-deleted mutants (PLC-γ1ΔSH22PC12), and SH3-deleted mutants (PLC-γ1ΔSH3PC12). Overexpressed whole PLC-γ1 or the SH2-SH2-SH3 domain of PLC-γ1 stimulated cell growth and inhibited NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-γ1 lacking the SH2-SH2 domain or the SH3 domain had no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-γ1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-γ1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-γ1 as well as intact PLC-γ1 could be tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-γ1 but to the SH2-SH2-SH3 domain of PLC-γ1.     

Interferon-β Is a Potent Promoter of Nerve Growth Factor Production by Astrocytes

Abstract: Recent clinical evidence has suggested that interferon-β is efficacious in the treatment of the demyelinating disease, multiple sclerosis. The mechanism of its efficacy remains unclear, and suggested modes of action have focused on immune modulation. Nonimmune effects of interferon-β may also contribute to its efficacy. Given that astrocytes produce a range of neurotrophic factors, we examined the possibility that interferon-β could increase the astrocytic production of nerve growth factor (NGF), which has been reported to cause oligodendrocytes to proliferate and to extend their processes; these phenotypes can impact favorably on remyelination. When the recombinant form of mouse interferon-β was added to mouse astrocyte cultures, a dose-dependent increase in NGF mRNA was obtained. The 40-fold increase in NGF mRNA elicited by 1,000 U/ml interferon-β was far more potent than that produced by other NGF-elevating agents in this study. In concordance, the protein for NGF was elevated by interferon-β. The production of NGF by interferon-β may be relevant to its clinical efficacy in multiple sclerosis. Furthermore, we suggest the potential utility of interferon-β in Alzheimer's disease.     

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.     

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

To define the molecular bases of growth factor-induced signal transduction pathways, antibodies known to block the activity of either protein kinase C (PKC) or the fos protein were introduced into PC12 cells by microinjection. The antibody against PKC significantly inhibited neurite outgrowth when scored 24 h after microinjection and exposure to nerve growth factor (NGF). Microinjection of antibodies to fos significantly increased the percentage of neurite-bearing cells after exposure to either NGF or basic fibroblast growth factor (bFGF) but inhibited the stimulation of DNA synthesis by serum, suggesting that in PC12 cells, fos is involved in cellular proliferation. Thus, activation of PKC is involved in the induction of neurite outgrowth by NGF, but expression of the fos protein, which is induced by both NGF and bFGF, is not necessary and inhibits neurite outgrowth.     

Effects of γ-IFN and NGF on subpopulations in a human neuroblastoma cell line: Flow cytometric and morphological analysis

Summary Neuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with γ-interferon (γ-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or-associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN +NGF treatment. These were significant increases in expression of all three proteins distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2–3 d, while those induced by IFN±NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4–6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with γ-IFN±NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.