Olga N. Chernyshev

Activated human microglia produce the excitotoxin quinolinic acid

WE aimed to determine the relative role of quinolinic acid synthesis in purified human microglia, monocytederived macrophages and astrocytes in the human brain following immune stimulation. Microglia and macrophages significantly increased quinolinic acid synthesis from tryptophan following activation by either lipopolysaccharide or interferon-γ. Quinolinic acid synthesis by individual microglia was heterogeneous, and its production by activated macrophages was approximately 32-fold greater than its microglial synthesis. Quinolinic acid synthesis by astrocytes was undetectable. Microglia may, therefore, be the primary endogenous cell type responsible for quinolinic acid synthesis in the brain parenchyma. However, under pathological conditions which precipitate blood–brain barrier compromise and/or leukocytic infiltration, intracerebral quinolinic acid may be derived chiefly from cells of the peripheral immune system such as activated macrophages.

Induction of Superoxide Anion and Nitric Oxide Production in Cultured Microgliaa

Although it is clear that the central nervous system (CNS) can undergo damage by exposure to reactive oxidant species (ROS), the source of the damaging molecules is not always apparent. The CNS, like other tissues, contains a variety of cellular mechanisms that are capable of generating both reactive oxygen and reactive nitrogen species, including autooxidation of molecules such as nor-epinephrine and dopamine as well as the action of specific enzymes such as xanthine oxidase or nitric oxide ~ynthase .~J .~-~ However, one potentially important source of ROS has been generally overlooked, that is, the production of ROS by the CNS macrophage, the microglia. Microglia were originally described by del Rio Hortega6 in 1932 and were correctly identified at that time as a CNS-specific component of the reticule endothelial system of the body. Like other tissue macrophages, the microglia generate superoxide anion and nitric oxide as part of their normal function. Nitric oxide is produced by the action of a cytosolic enzyme known as the inducible nitric oxide synthase (iNOS), while superoxide anion is produced by a membrane-bound NADPH o x i d a ~ e . ~ * ~ . ~ ~ The release of these reactive oxidants from microglia can be important to the CNS for two primary reasons: (1) microglia are distributed in a matrixlike arrangement throughout all regions of the CNS,9.l0 and (2) the generation of reactive oxidants by microglia can be induced under a variety of conditions, including injury and i n f l a m m a t i ~ n . ~ ~ ~ ~ Microglia, therefore, not only generate significant quantities of reactive oxidants but are also in an ideal position to promote oxidative damage to neurons and other glia. In order to understand how microglia contribute to changes in the oxidative environment of the CNS, we have examined the regulation of superoxide anion and nitric oxide production by cultured microglia. Microglia were prepared from a mixed glial culture of neonatal rat cerebral cortices as previously de~cr ibed .~ lJ~ Cells were grown at 37°C in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1 mM glutamine, and 25 pg/ml gentamycin for 14 days. On day 14, the microglia were separated from the mixed glial culture by shaking, the

Microglial Contribution to Oxidative Stress in Alzheimer's Disease

Abstract: Microglia are the CNS macrophage and are a primary cellular component of plaques in Alzheimers disease (AD) that may contribute to the oxidative stress associated with chronic neurodegeneration. We now report that superoxide anion production in microglia or macrophages from 3 different species is increased by long term exposure (24 hours) to Aβ peptides. Since Aβ competes for the uptake of opsonized latex beads and for the production of superoxide anion by opsonized zymosan, a likely site of action are membrane receptors associated with the uptake of opsonized particles or fibers. The neurotoxic fibrillar peptides Aβ (1–42) and human amylin increase radical production whereas a non‐toxic, non‐fibrillar peptide, rat amylin, does not. We also report that the effect of Aβ peptides on superoxide anion production is not associated with a concomitant increase in nitric oxide (NO) production in either human monocyte derived macrophages (MDM) or hamster microglia from primary cultures. Since NO is known to protect membrane lipids and scavenge superoxide anion, the lack of Aβ‐mediated induction of NO production in human microglia and macrophages may be as deleterious as the over‐production of superoxide anion induced by chronic exposure to Aβ peptides.

INHIBITION OF MICROGLIAL SUPEROXIDE ANION PRODUCTION BY ISOPROTERENOL AND DEXAMETHASONE

Microglia, like other tissue macrophages, are a component of the hypothalamic-pituitary endocrine-immune axis and, as such, are responsive to both neural and endocrine factors. Using cultured neonatal hamster microglia, we have examined the effect of isoproterenol, a beta-adrenergic agonist, and dexamethasone, a synthetic glucocorticoid, on superoxide anion production. For these experiments, microglia were pretreated with isoproterenol or dexamethasone and then induced to produce superoxide anion by exposure of the cells to phorbol myristate acetate (PMA). Our study demonstrates that the PMA-stimulated production of superoxide anion was decreased by acute (30 min) and chronic (24 h) pretreatment of the microglia with isoproterenol and was blocked by the beta-adrenergic receptor antagonist, propranolol. Since a rise in intracellular cAMP may be a prime factor in the inhibition of superoxide anion production in isoproterenol-treated cells, we used forskolin, a known activator of the adenylate cyclase in place of isoproterenol and re-investigate superoxide anion production. Short term exposures to forskolin produced a lower amount of superoxide anion than PMA-stimulated alone and, thus, mimicked the effect of isoproterenol. However, treatment with the same concentration of forskolin for 24 h prior to the induction of the NADPH oxidase did not significantly change PMA-stimulated superoxide anion production from untreated values. Thus, chronic exposure to forskolin produced a different effect than chronic exposure to isoproterenol. Isoproterenol and forskolin both increased immunoreactivity for the protein products of the early response genes, c-fos and c-jun. Pretreatment with dexamethasone for 24 h also inhibited superoxide anion production and was blocked by the protein synthesis inhibitor, cycloheximide. The simultaneous addition of varying concentrations of dexamethasone and 5 microM isoproterenol did not produce a greater inhibition in superoxide anion production than either agent alone. The down-regulation of microglial function by adrenergic agonists and by glucocorticoids provides a way in which the cytotoxicity of these immune cells can be reduced and may be a factor in the paracrine regulation of microglia.

Polyribonucleotides induce nitric oxide production by human monocyte-derived macrophages.

Cytokine‐mediated activation of inducible nitric oxide synthase (iNOS) in monocytes or macrophages is species specific. In contrast to rat or mouse, human macrophages do not produce measurable levels of nitric oxide (NO) when induced by inflammatory mediators. Exposure to noncytokine mediators such as tumor cells or viruses, however, has recently been shown to activate human iNOS. NO production in response to these mediators is much lower than that seen for rat or mouse cells and often requires several days of stimulation. We have found that the synthetic, double‐stranded polyribonucleotide polyinosinic‐polycytidilic acid (Poly I:C), commonly used to mimic viral exposure, activated iNOS in human monocyte‐derived macrophages (MDM). The production of NO, measured by nitrite accumulation, was detected after 24 h of stimulation with Poly I:C. The single‐stranded polyribonucleotide Poly I, but not Poly C, also increased NO production. Nitrite production was enhanced when the MDM were primed (pretreated) with γ or α interferon or other immune mediators such as IL‐4 and was reduced by the iNOS inhibitor, N‐methyl‐L‐arginine (L‐NMMA). The use of Poly I:C to initiate NO production in human macrophages provides a useful tool to study the differences between the commonly used animal models and human cells and may provide insight into the pathophysiological significance of these differences. J. Leukoc. Biol. 62: 369–373; 1997.

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport

Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.

BIOLOGICAL ACTIVITY OF INTERLEUKIN-10 IN THE CENTRAL NERVOUS SYSTEM

Cytokines play a crucial role as mediators of inflammation. Astrocytes and microglia are the two major glial cells involved in the central nervous system immune responses. In this study we examined the effects of interleukin-10 (IL-10), one of the naturally occurring inhibitory cytokines, on different types of glial cells in culture such as rat astrocytes, hamster microglia and C6-2B glioma cells. Phosphorylation of signal transducers and activators of transcription (STAT) proteins was used as a marker for IL-10 activity. Within minutes, IL-10 elicited a strong and weak increase in STAT3 and STAT1 phosphorylation, respectively, in human T lymphocytes, suggesting that STAT3 is a main IL-10 signaling event in these cells. In contrast, IL-10 failed to induce STAT3 in glial cells, but elicited a weak increase in STAT1 phosphorylation in microglia and C6-2B glioma cells only, suggesting that in some glial cell population(s) IL-10 may produce cellular responses via activation of the STAT1 pathway. Moreover, in C6-2B cells, IL-10 elicited a decrease in the level of basic fibroblast growth factor mRNA. A similar decrease was observed in adult rat hypothalamus, indicating that this cytokine may regulate glial production of trophic factors. Our data suggest that IL-10 may play a role in glial cell differentiation and proliferation.

COMMENTARYINHIBITION OF MICROGLIAL SUPEROXIDE ANION PRODUCTION BY ISOPROTERENOL AND DEXAMETHASONE

Microglia, like other tissue macrophages, are a component of the hypothalamic-pituitary endocrine-immune axis and, as such, are responsive to both neural and endocrine factors. Using cultured neonatal hamster microglia, we have examined the effect of isoproterenol, a beta-adrenergic agonist, and dexamethasone, a synthetic glucocorticoid, on superoxide anion production. For these experiments, microglia were pretreated with isoproterenol or dexamethasone and then induced to produce superoxide anion by exposure of the cells to phorbol myristate acetate (PMA). Our study demonstrates that the PMA-stimulated production of superoxide anion was decreased by acute (30 min) and chronic (24 h) pretreatment of the microglia with isoproterenol and was blocked by the beta-adrenergic receptor antagonist, propranolol. Since a rise in intracellular cAMP may be a prime factor in the inhibition of superoxide anion production in isoproterenol-treated cells, we used forskolin, a known activator of the adenylate cyclase in place of isoproterenol and re-investigate superoxide anion production. Short term exposures to forskolin produced a lower amount of superoxide anion than PMA-stimulated alone and, thus, mimicked the effect of isoproterenol. However, treatment with the same concentration of forskolin for 24 h prior to the induction of the NADPH oxidase did not significantly change PMA-stimulated superoxide anion production from untreated values. Thus, chronic exposure to forskolin produced a different effect than chronic exposure to isoproterenol. Isoproterenol and forskolin both increased immunoreactivity for the protein products of the early response genes, c-fos and c-jun. Pretreatment with dexamethasone for 24 h also inhibited superoxide anion production and was blocked by the protein synthesis inhibitor, cycloheximide. The simultaneous addition of varying concentrations of dexamethasone and 5 microM isoproterenol did not produce a greater inhibition in superoxide anion production than either agent alone. The down-regulation of microglial function by adrenergic agonists and by glucocorticoids provides a way in which the cytotoxicity of these immune cells can be reduced and may be a factor in the paracrine regulation of microglia.