Jean E. Merrill

Cytokines in inflammatory brain lesions: helpful and harmful

Multiple sclerosis (MS) is thought to be an autoimmune disease. In healthy individuals, the T cells of the immune system, when activated by an infectious agent, regularly traffic across an intact blood-brain barrier, survey the CNS and then leave. In MS, for reasons that are only gradually being understood, certain events in the peripheral immune response and in the brain cause some autoreactive T cells to stay in the CNS. Their presence initiates infiltration by other leukocytes and activation and recruitment of endogenous glia to the inflammatory process, ultimately leading to the destruction of myelin and the myelin-producing cell, the oligodendrocyte, and the dysfunction of axons. The key mediators in the subsequent cycles of histological damage and repair, and clinical relapse and remission are thought to be adhesion molecules, chemokines and cytokines.

Intercellular signaling in glial cells: Calcium waves and oscillations in response to mechanical stimulation and glutamate

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.

Tumor necrosis factor alpha, interleukin 1 and related cytokines in brain development: normal and pathological.

Microglia and astrocytes produce several cytokines including interleukin 1 (IL1) and tumor necrosis factor alpha (TNF alpha), which have pleiotropic effects in the immune and nervous systems. Recent evidence has come to light that they play a role in damage in the central nervous system. This indeed may be the result of overproduction of these factors as the consequence of trauma or disease. At lower concentrations, however, they may in fact be involved in the normal development of the nervous system. A review of brain IL1 and TNF alpha during normal development, abnormal development, and pathology is presented here. We have speculated as to the association of these cytokines directly and indirectly with neural cell migration, proliferation, differentiation, and death.

Nitric oxide as a potential pathological mechanism in demyelination: Its differential effects on primary glial cellsin vitro

Because we believe that macrophage-derived nitric oxide contributes to pathology of demyelinating diseases, we have determined the differential effects of nitric oxide on primary rat glial cells in vitro. Enriched cultures of microglia, astrocytes and oligodendrocytes were treated with S-nitroso,N-acetyl-DL-penicillamine, a nitric oxide-releasing chemical. There was a significantly decreased function of one of the ferrosulfur-containing mitochondrial enzymes after S-nitroso,N-acetyl-DL-penicillamine/nitric oxide treatment in oligodendrocytes and astrocytes compared to microglia, which were much less sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide at all concentrations. At 0.5 mM S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, astrocytes and oligodendrocytes suffered a 40% loss in succinate dehydrogenase activity, while microglia were unaffected. A control non-ferrosulfur-containing mitochondrial enzyme, isocitrate dehydrogenase, was not affected in any glial cell type. Although the per cent of mitochondrial damage in oligodendrocytes and astrocytes was the same for all concentrations of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, significant cell death occurred in oligodendrocytes at 1.0 mM; at this concentration there was no significant killing of microglia or astrocytes. Furthermore, at a 0.5 mM concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, which inhibited mitochondrial respiration but did not kill oligodendrocytes, significant changes in oligodendrocyte morphology (e.g. retraction of processes) occurred. Morphological changes were not seen in microglia and astrocytes at any concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide. In addition, oligodendrocytes were more sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide-induced single stranded DNA breaks than microglia or astrocytes. The mitochondrial damage was attributable to nitric oxide since N-acetyl-DL-penicillamine had no effect. Oxyhemoglobin, which competitively inhibits toxic effects of nitric oxide, protected these glial cells from mitochondrial damage, single stranded breaks in DNA and cell death in a time- and dose-dependent manner. Once again, oligodendrocytes were less easily rescued from nitric oxide effects by oxyhemoglobin than were astrocytes, suggesting greater vulnerability of the myelin-producing cell to nitric oxide. These findings suggest that there is differential sensitivity of glial cells to nitric oxide. Although oligodendrocytes and astrocytes are equally susceptible to nitric oxide-induced mitochondrial damage, oligodendrocytes are more sensitive to nitric oxide-induced single stranded DNA breaks, morphological changes and cell death. Compared to both oligodendrocytes and astrocytes, microglia, nitric oxide-producing cells, are resistant to nitric oxide-induced damage.

Differential expression, cytokine modulation, and specific functions of type-1 and type-2 tumor necrosis factor receptors in rat glia

Tumor necrosis factor alpha (TNF alpha) and lymphotoxin alpha (LT alpha) induce pleiotropic cellular effects through low-affinity 55 kDa type-1 receptors (TNFR1, CD120a) and high-affinity 75 kDa type-2 receptors (TNFR2, CD120b). Both cytokines have potent biological effects on glial cells and are strongly implicated in the pathology of central nervous system (CNS) demyelinating diseases. However, to date, neither constitutive nor cytokine-induced TNFR expression by glial cells have been definitively characterized. We therefore characterized TNF receptors at the molecular, protein, and functional levels in rat astrocytes, microglia, and oligodendrocytes. Northern blotting demonstrated that all three types of glia constitutively transcribed a single TNFR1 mRNA. IFN gamma increased transcript levels in all three types of glia, but TNF alpha increased levels only in oligodendrocytes Microglia constitutively transcribed three distinct TNFR2 mRNAs, levels of which were increased by either IFN gamma or TNF alpha. In contrast, astrocytes and oligodendrocytes constitutively transcribed nearly undetectable levels of TNFR2 mRNAs, and levels were not affected by IFN gamma, TNF alpha, or oligodendrocyte maturation. Immunocytochemical staining of glial cells corroborated Northern data by demonstrating that glia express a parallel pattern of TNFR proteins on their cell surfaces. In co-cultures of microglia plated atop irradiated astrocytes, human TNF alpha (which, on mouse cells, binds TNFR1 exclusively) induced microglial cell proliferation, whereas murine TNF alpha (which binds both TNFRs) did not. Collectively, the data show that microglia, a primary source of TNF alpha at CNS inflammatory sites, express both TNFR1 and TNFR2, whereas astrocytes and oligodendrocytes, whose embryological origin differs from that of microglia, predominantly express TNFR1. TNF alpha increases expression of TNFR1 by oligodendrocytes whereas it increases expression of TNFR2 by microglia. Microglia proliferation data suggest that signals transduced through TNFR2 directly or indirectly inhibit signals transduced through TNFR1. Different patterns of TNFR expression by glia at sites of CNS inflammation may be critical in determining whether TNF has activational, proliferative, or cytotoxic effects on these cells.

Effects of Interleukin-1 and Tumor Necrosis Factor-α on Astrocytes, Microglia, Oligodendrocytes, and Glial Precursors in vitro

Microglia and astrocytes undergo proliferative and differentiative changes in vivo after trauma or diseases such as multiple sclerosis (MS). Oligodendrocytes are destroyed in lesions in MS. Interleukin-1 (IL1) and tumor necrosis factor-alpha (TNF alpha) are involved in inflammation of the central nervous system and are elevated in MS. We have investigated the changes in cell morphology and cell number induced by IL1 and TNF alpha in purified and mixed populations of primary rat brain microglia, astrocytes, oligodendrocytes, and glial precursors. Depending on the target population, proliferation, differentiation, or inhibition of cultured cells was observed. The data also suggest that interactions among cell populations occur and support the hypothesis that IL1 and TNF alpha effects may be indirect, possibly through induction of other factors.

Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes

We have investigated the mechanism of nitric oxide-induced damage in glial cells. Genomic DNA isolated from astrocytes and microglia, treated for 18 h with varying concentrations of a nitric oxide donor, was analysed by electrophoresis. No DNA damage was evident. Oligodendrocytes, treated with 2 mM nitric oxide for 3-48 h, showed single stranded breaks at 48 h but no laddering of nucleosomic fragments of DNA. When analysed by electron microscopy, ultrastructural changes in oligodendrocytes treated with 1 mM nitric oxide for 24 h showed intact nuclei but alterations in membranes and organelles characteristic of necrosis, including disrupted mitochondria with dissolution of their christae. Astrocytes, a glial cell type that we have previously shown to be much less sensitive to nitric oxide-induced damage, did not show ultrastructural changes. DNA analysis by flow cytometry of glial cells treated with nitric oxide supported the apparent necrotic-type death in oligodendrocytes. Double staining of oligodendrocytes, using Hoechst 33342 and propidium iodide for the simultaneous assessment of both apoptotic and necrotic cells, demonstrated that, while the proportion of dead cells increased with time and increasing concentrations of nitric oxide, the death was due to necrosis and not apoptosis. In this study, we demonstrate that direct exposure to soluble nitric oxide, produced in vitro from a nitric oxide donor chemical, ultimately kills oligodendrocytes by necrosis. Microglia and astrocytes maintain DNA and organelle integrity when exposed to exogenous nitric oxide.

The role of nitric oxide in multiple sclerosis.

Abstract During the past decade nitric oxide has emerged as an important mediator of physiological and pathophysiological processes. Elevated nitric oxide biosynthesis has been associated with nonspecific immune-mediated cellular cytotoxicity and the pathogenesis of chronic, inflammatory autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, and mutiple sclerosis. Recent evidence suggests, however, that nitric oxide is also immunoregulatory and suppresses the function of activated proinflammatory macrophages and T lymphocytes involved in these diseases. This article reviews the role of nitric oxide in the biology of central nervous system glial cells (astrocytes and microglia) as it pertains to the pathogenesis of multiple sclerosis in humans and experimental allergic encephalitis, the animal model of this disease. Although nitric oxide has been clearly implicated as a potential mediator of microglia-dependent primary demyelination, a hallmark of multiple sclerosis, studies with nitric oxide synthase inhibitors in the encephalitis model have been equivocal. These data are critically reviewed in the context of what is know from clinical research on the nitric oxide pathway in multiple sclerosis. Specific recommendations for future preclinical animal model research and clinical research on the nitric oxide pathway in patients are suggested. These studies are necessary to further define the role of nitric oxide in the pathology of multiple sclerosis and to fully explore the potential for nitric oxide synthase inhibitors as novel therapeutics for this disease.

Inducible Nitric-oxide Synthase and Nitric Oxide Production in Human Fetal Astrocytes and Microglia A KINETIC ANALYSIS

The understanding of the induction and regulation of inducible nitric-oxide synthase (iNOS) in human cells may be important in developing therapeutic interventions for inflammatory diseases. In the present study, we not only demonstrated that human fetal mixed glial cultures, as well as enriched microglial cultures, synthesize iNOS and nitric oxide (NO) in response to cytokine stimulation, but also assessed the kinetics of iNOS and NO synthesis in human fetal mixed glial cultures. The iNOS mRNA was expressed within 2 h after stimulation and decreased to base line by 2 days. Significant levels of iNOS protein appeared within 24 h after stimulation and remained elevated during the culture period. A dramatic increase in NO production and NO-mediated events, such as the induction of cyclic guanosine monophosphate (cGMP), NADPH diaphorase activity, and nitrotyrosine occurred 3 days after stimulation, a delay of 48 h from the time of the first expression of iNOS enzyme. This delay of NO production was altered by the addition of tetrahydrobiopterin, but not by the addition of L-arginine, heme, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), or NADPH. These findings suggest that a post-translational regulatory event might be involved in iNOS-mediated NO production in human glia.

Remyelination by oligodendrocytes stimulated by antiserum to spinal cord

The new synthesis of myelin and the proliferation of oligodendrocytes was stimulated by serum from syngeneic mice immunized with homogenized spinal cord (SCH). Treatment with this antiserum produced a 10-fold increase in the area of remyelination in spinal cords that had become demyelinated previously as a result of infection by Theilers murine encephalomyelitis virus. Inflammation was decreased in regions of white matter that showed remyelination. Oligodendrocytes exposed to anti-SCH in vitro incorporated three to five times more [PH]thymidine than resting cells did and expressed more myelin basic protein in their cytoplasm, suggesting stimulation of myelinogenesis. Thus, there is a factor present in anti-SCH antiserum that stimulates central nervous system-type remyelination. This finding may provide clues for the therapy of patients with demyelinating disorders such as multiple sclerosis.