Giulio Levi

Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide

Microglial cells are believed to play an active role in brain inflammatory, immune and degenerative processes. Depending on the magnitude of microglial reaction, on the type of stimulus and on the concurrence of other local factors, microglia can contribute to host defence and repair, or to the establishment and maintenance of brain damage. Many of the effects of microglial cells can be ascribed to the numerous substances that these cells can synthesize and release in response to a variety of stimuli (cytokines, pro-inflammatory substances, neurotransmitters, toxins, etc.). The present article deals with two classes of compounds that activated microglial cells can produce in large amounts: prostanoids (that derive from arachidonic acid through the cyclooxygenase pathway), and nitric oxide (that is synthesized from arginine by nitric oxide synthase). Prostanoids and nitric oxide have a number of common targets, on which they may exert similar or opposite actions, and have a crucial role in the regulation of inflammation, immune responses and cell viability. Their synthesis can massively increase when the inducible isoforms of cyclooxygenase and nitric oxide synthase are expressed. The metabolic pathways of prostanoids and nitric oxide are finely tuned by the respective end-products, by cyclic AMP and by a number of exogenous factors, such as cytokines, glucocorticoids, lipocortin-1 and others. Some of these factors (e.g. transforming growth factor-beta 1, interleukin-10, lipocortin-1) may be secreted by microglial cells themselves, and act in an autocrine-paracrine way. In view of the neuroprotective role attributed to some prostaglandins and to the cytotoxicity of excessive levels of nitric oxide or its derivatives, the balance between prostanoid and nitric oxide levels may be crucial for orienting microglial reactions towards neuroprotection or neurotoxicity.

A simple apparatus for studying the release of neurotransmitters from synaptosomes

Abstract A simple superfusion technique for studying the release of neurotransmitters from preloaded synaptosomes is described. As compared with previously described procedures the method has the following advantages: (1) prevention of re-uptake; (2) immediate utilization of preloaded synaptosomes; (3) great flexibility and reliability; (4) continuous determination of release from parallel samples.

Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures.

Granule cells from 8-day-old rat cerebella were grown in basal Eagles medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity greater than 90%. The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog [3H]D-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled. In contrast, astrocytes were heavily labeled at all stages. (2) Calcium-dependent, high [K+]-induced release, or tetrodotoxin-sensitive, veratridine-induced release of [3H]D-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV. (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release [3H]D-aspartate and endogenous glutamate almost unchanged. (4) Newly synthesized [3H]glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with [3H]D-aspartate), but was specifically released from neuronal structures (perikarya and processes) by depolarizing stimuli.

Carrier-mediated release of neurotransmitters

There is growing evidence that neurotransmitters can be released not only by exocytosis but also through the membrane carriers responsible for transmitter reuptake. Giulio Levi and Maurizio Raiteri review the in vitro and in vivo evidence supporting the existence of a carrier-mediated release for different classes of transmitters. While the physiological significance of carrier-mediated release remains speculative, widely used drugs such as sympathomimetic amines, the anorectic drug fenfluramine and some drugs of abuse act in part by stimulating monoamine carrier-mediated release. Moreover, antidepressants known to inhibit monoamine reuptake, can block carrier-mediated release. This mechanism may also come into play in pathological conditions such as ischaemia.

Role of the peroxisome proliferator-activated receptor-γ (PPAR-γ) and its natural ligand 15-deoxy-Δ12,14-prostaglandin J2 in the regulation of microglial functions

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of a large group of nuclear receptors controlling the proliferation of peroxisomes that is involved in the downregulation of macrophage functions. Here, we report that PPAR‐γ was constitutively expressed in rat primary microglial cultures and that such expression was downregulated during microglial activation by endotoxin (LPS). The presence of the PPAR‐γ natural ligand 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) counteracted the repression of PPAR‐γ expression caused by LPS. In microglial cultures stimulated by LPS, interferon‐γ (IFN‐γ) or by their combination, 15d‐PGJ2 reduced the production of nitric oxide (NO) and the expression of inducible NO synthase (iNOS). The inhibitory effect was dose‐dependent and did not involve an elevation of cyclic AMP, a second messenger known to inhibit NOS expression in microglia. In addition, 15d‐PGJ2 down‐regulated other microglial functions, such as tumour necrosis factor‐α (TNF‐α) synthesis and major histocompatibility complex class II (MHC class II) expression. The effects of 15d‐PGJ2 occurred, at least in part, through the repression of two important transcription factors, the signal transducer and activator of transcription 1 and the nuclear factor κB, known to mediate IFN‐γ and LPS cell signalling. Our observations suggest that 15d‐PGJ2, the synthesis of which is likely to occur within the brain, could play an important role in preventing brain damage associated with excessive microglial activation.

Effect of sympathomimetic amines on the synaptosomal transport of noradrenaline, dopamine and 5-hydroxytryptamine.

The interaction of sympathomimetic amines with the transport of 3H-noradrenaline (3H-NE), 3H-dopamine (3H-DA) and 3H-5-hydroxytryptamine (3H-5-HT) were investigated in rat hypothalamic (3H-NE) and striatal (3H-DA) and 3 H-5-HT) synaptosomes. Modifications in the phenylethylamine structure led to changes in activity towards biogenic amine uptake and release: (a) the introduction of a beta-OH group led to compounds less active in inhibiting uptake and stimulating release of 3H-NE, 3H-DA and 3H-5-HT, with the exception of 3H-NE release which was stimulated more by unlabeled 1-NE than by DA; (b) the introduction of phenolic-OH groups always led to compounds which were stronger uptake inhibitors and releasers of the three biogenic amines; (c) the alpha-methylation increased the potency towards uptake inhibition and release stimulation, with the exception of 3H-NE release: in fact, the releasing activity of phenylethylamine was suppressed by alpha-methylation; (d) the introduction of a -Cl group in the para position selectively potentiated the effects on 3H-5-HT uptake and release and generally depressed those on catecholamine transport.

The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions

B7 is a constimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferom B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-I, LFA-I and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimers disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions.

Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures.

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose‐ and time‐dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100‐fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β‐adrenergic receptor activation. The β‐agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide‐evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β‐adrenergic activation.

d-Amphetamine as a releaser or reuptake inhibitor of biogenic amines in synaptosomes.

The effect of d-amphetamine on the release of tritiated norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) was analyzed in synaptosomes from different brain area. 3H-NE release was unaffected in the hypothalamus, a region which is rich in noradrenergic terminals, and in cerebellum and pons-medulla, but was substantially increased in corpus striatum and moderately in cerebral cortex. 3H-DA release was strongly enhanced in corpus striatum, a region rich in dopaminergic terminals, substantially increased in cerebral cortex, and slightly increased in the hypothalamus. Since the regional pattern of d-amphetamine-stimulated release was similar with the two catecholamines, but the stimulation was greater with 3H-DA than with 3H-NE, and was more evident in areas richer in dopaminergic terminals, it is suggested that the drug can release 3H-DA or artificially stored 3H-NE from dopaminergic terminals, but not 3H-NE, from noradrenergic terminals. d-Amphetamine also seems capable of releasing 3H-5-HT from serotoninergic terminals. In contrast with the two catecholamines, 3H-5-HT release was more enhanced in cerebral cortex than in corpus striatum.

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.