Giuliana M. Lauro

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor α (TNFα)-inducible NO synthesis, because by affecting NF-κB activation it inhibits inducible Ca-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNFα-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNFα induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNFα-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNFα-elicited NF-κB activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-κB availability.

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid beta (Abeta) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt-Jakob disease, while Abeta is involved in the pathogenesis of Alzheimers disease. Synthetic fragments of both proteins, PrP106-126 and beta25-35 (beta25-35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106-126 and beta25-35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca2+]i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca2+]i concentration as observed after treatment with PrP106-126 and beta25-35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106-126 and beta25-35.

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimers disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression.

S100B protects LAN‐5 neuroblastoma cells against Aβ amyloid‐induced neurotoxicity via RAGE engagement at low doses but increases Aβ amyloid neurotoxicity at high doses

At the concentrations normally found in the brain extracellular space the glial‐derived protein, S100B, protects neurons against neurotoxic agents by interacting with the receptor for advanced glycation end products (RAGE). It is known that at relatively high concentrations S100B is neurotoxic causing neuronal death via excessive stimulation of RAGE. S100B is detected within senile plaques in Alzheimers disease, where its role is unknown. The present study was undertaken to evaluate a putative neuroprotective role of S100B against Aβ amyloid‐induced neurotoxicity. We treated LAN‐5 neuroblastoma cultures with toxic amounts of Aβ25‐35 amyloid peptide. Our results show that at nanomolar concentrations S100B protects cells against Aβ‐mediated cytotoxicity, as assessed by 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP‐fluorescein isothiocyanate nick end‐labeling (TUNEL) experiments, by countering the Aβ‐mediated decrease in the expression of the anti‐apoptotic factor Bcl‐2. This effect depends on S100B binding to RAGE because S100B is unable to contrast Aβ‐mediated neurotoxicity in neurons overexpressing a signaling‐deficient RAGE mutant lacking the cytosolic and transducing domain. Our data suggest that at nanomolar doses S100B counteracts Aβ peptide neurotoxicity in a RAGE‐mediated manner. However, at micromolar doses S100B is toxic to LAN‐5 cells and its toxicity adds to that of the Aβ peptide, suggesting that additional molecular mechanisms may be involved in the neurotoxic process.

Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor α stimulation

This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

The role of the L‐arginine‐nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) was investigated. Incubation of T 67 astroglial cell line with IL‐β (10 ng ml−1) and TNF‐α (500 u ml−1) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 20–300 μM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine‐treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL‐1β and TNF‐α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μM). The inhibition elicited by L‐NAME on PGE2‐release by cytokine‐treated astroglial cells was reversed by adding AA (40 μM), showing that the effect of NO on cytokine‐dependent PGE2 release occurred at the cyclo‐oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine‐treated astroglial cells was inhibited by indomethacin (10 μM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX‐2) was involved. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine‐treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL‐1β and TNF‐α is due to enhanced COX‐2 activity via activation of the L‐arginine‐NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

PLATELET-ACTIVATING FACTOR PRODUCTION BY HUMAN FETAL MICROGLIA: EFFECT OF LIPOPOLYSACCHARIDES AND TUMOR NECROSIS FACTOR-ALPHA

Since platelet-activating factor (PAF) exerts neurotoxic effects on brain cells, we explored the possibility of PAF production by human fetal microglial cells in vitro. PAF content in pure cultures was assayed and characterized in basic conditions, and after stimulation with growth factors and cytokines. Results showed that microglia cells synthesized PAF when challenged with tumor necrosis factor-alpha and lipopolysaccharides, whereas other molecules, such as gamma-interferon or basic fibroblast growth factor, were ineffective. The induced PAF production was concentration- and time-dependent. These results are in line with the hypothesis that microglia can start a cascade of events leading to tissue damage, thus playing a central role in the pathogenesis of several central nervous system diseases.

Bacterial Lipopolysaccharide Plus Interferon-γ Elicit a Very Fast Inhibition of a Ca2+-dependent Nitric-oxide Synthase Activity in Human Astrocytoma Cells

Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-γ, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-γ (MIX) strongly inhibits the cNOS activity, as determined by measuring [3H]citrulline production. In addition, the effect of MIX is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MIX does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction.

Inhibition of inducible nitric oxide synthase mRNA expression by basic fibroblast growth factor in human microglial cells

The effect of basic fibroblast growth factor (bFGF) on inducible nitric oxide synthase (iNOS) mRNA expression in human cultured ramified microglial cells was investigated. Using RT-PCR and Southern blot analysis, we found that bFGF prevented the iNOS gene expression as induced by LPS/TNF alpha. Also, bFGF dose-dependently inhibited nitrite levels in treated cell supernatants. That the early presence of bFGF during LPS/TNF alpha induction was essential indicates that iNOS gene expression can be transcriptionally regulated.

Interferon gamma up-regulates α2 macroglobulin expression in human astrocytoma cells

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor alpha 2 macroglobulin (alpha 2M). Interferon gamma (IFN gamma), a potent immunoregulatory lymphokine, was able to increase the synthesis of alpha 2M by these cells, as measured by ELISA on cell supernatants. The alpha 2M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures following IFN gamma treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFN gamma. A corresponding enhanced alpha 2M mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of alpha 2M during inflammatory and immune processes in the CNS. An increased release of alpha 2M following IFN gamma stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes.