Yuen Ling Lee

Tumor Necrosis Factor‐α and Basic Fibroblast Growth Factor Decrease Glial Fibrillary Acidic Protein and Its Encoding mRNA in Astrocyte Cultures and Glioblastoma Cells

Abstract: Tumor necrosis factor‐α is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor‐α. Treatment with tumor necrosis factor‐α for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor‐α treatment increased the expression of the cytokine interleukin‐6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor‐α, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor‐α induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor‐α. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor‐α treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.

Effects of Pulsed Magnetic Stimulation on GFAP Levels in Cultured Astrocytes

The present study evaluates the physiological effects of magnetic stimulation on astrocyte cultures. Cell cultures were exposed to pulsed magnetic stimulation (10 Hz, 10 sec) at the following levels: 0.10 tesla (T; Group A); 0.21 T (Group B); 0.42 T (Group C); and 0.63 T (Group D). Glial fibrillary acidic protein (GFAP) levels from immunoblots, total protein concentrations, and cellular morphology were analyzed at 0, 1, 3, 5, 7, 13, and 20 days poststimulation. Significantly higher GFAP levels were observed in Group D at day 3 (P = 0.0114). The change was transient as the GFAP levels quickly returned to control levels by day 5. No other significant changes in GFAP levels were observed. In comparison to control protein levels at day 0, concentrations from Groups B, C, and D were significantly lower (P < 0.006), whereas at day 3, Groups C and D were significantly higher (P < 0.02). Differences in astrocyte morphology due to magnetic stimulation were not observed. This study demonstrated that high intensity magnetic stimulation for only 10 sec induced a transient biological response. J. Neurosci. Res. 55:238–244, 1999. Published 1999 Wiley‐Liss, Inc.

Leukemia inhibitory factor mRNA is expressed in cortical astrocyte cultures but not in an immortalized microglial cell line

Leukemia inhibitory factor (LIF) is a multifunctional cytokine synthesized by a variety of cell types. In the nervous system LIF affects neuronal differentiation, and may be important during cerebral infection and inflammation. To clarify the cellular source of LIF in the brain, we examined the expression of LIF mRNA by primary cortical astrocyte cultures and an immortalized microglial cell line. The microglial cell line did not express LIF mRNA in response to pro-inflammatory agents such as lipopolysaccharide (LPS) that induced expression of other cytokine mRNAs. In contrast, primary astrocyte cultures grown in serum-containing medium expressed LIF mRNA constitutively, and this expression was regulated by pro-inflammatory and anti-inflammatory stimuli. Agents which activate the cAMP and protein kinase C second messenger systems also increased LIF mRNA in astrocyte cultures. These results suggest that astrocytes, but not microglia, may be an important source of LIF during cerebral inflammation and infection.

Chapter 21: Glutamate as an energy substrate for neuronal-astrocytic interactions

Publisher Summary This chapter discusses the role of glutamate as a metabolic substrate. Glutamate is the most plentiful amino acid and the major excitatory neurotransmitter in adult central nervous system (CNS). Glutamate participates in the synthesis of proteins, peptides and fatty acids, and in the control of osmotic or anionic balance. It is a constituent of at least two important co-factors, glutathione and folic acid; it contributes along with glutamine to the regulation of ammonia levels, and it serves as precursor for GABA and various tricarboxylic acid (TCA) cycle intermediates. Glutamate is released from neurons in large amounts. Uptake studies demonstrates that both neurons and astrocytes take up glutamate. The uptake of glutamate into astrocytes represents a net transfer of carbon skeleton from the neurons to astrocytes. There are three probable roles for this uptake process: (1) to remove the glutamate from extracellular space and synaptic clefts as a means of termination of the transmitter activity; (2) to form glutamine during the detoxification of ammonia and (3) to serve as a metabolic substrate for astrocytes. Metabolic studies have shown that, a part of the glutamate taken up by astrocytes is metabolized to CO2 and another part to glutamine, the latter of which then can be returned to neurons as a precursor for glutamate and GABA. Glutamate exerts a regulatory effect on glycogen metabolism in astrocytes and also affects glucose utilization in astrocytes. Glutamate also plays an important role in the pathogenesis of various neurologic diseases and insult.

CALCIUM ION DEPENDENCE OF THE 2-SITE IMMUNORADIOMETRIC ASSAY OF MOORE'S S-100 PROTEIN

Abstract— The two‐site immunoradiometric assay (two‐site IRMA) for a specific protein of the nervous system, S‐100, is carried out by reaction of the S‐100 protein solution with a solid‐phase anti(S‐100) followed by a second reaction in which the insoluble product is incubated with purified, radioactive anti(S‐100). Unreacted labeled antibodies remain in solution and are washed away. As the amount of S‐100 increases, the radioactivity in the solid‐phase increases. The most significant assay variable is the effect of calcium on the assay dose‐response. 0.1 mM‐EDTA causes a total inhibition of the dose‐response curve which is reversed by increasing the concentration of calcium ions. The dose‐response reaches a maximum at 1.0mM‐Ca2+. then becomes progressively inhibited as the Ca2+ concentration is increased further. Previous immunochemical studies of S‐100 which did not allow for this effect must now be interpreted with caution.

Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response.

The receptor for macrophage colony‐stimulating factor (M‐CSFR; c‐fms) is expressed at increased levels by microglia in Alzheimers disease (AD) and in mouse models for AD. Increased expression of M‐CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M‐CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac‐1 integrin α subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c‐fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c‐fms construct resulted in expression of the c‐fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M‐CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)‐1α macrophage inflammatory protein (MIP‐1α), and trends toward increased production of IL‐6 and M‐CSF. These findings demonstrate that microglial overexpression of M‐CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M‐CSFR could contribute to the inflammatory response observed in AD brain.

Chapter 20 A RT-PCR study of gene expression in a mechanical injury model

Publisher Summary This chapter discusses the reverse transcriptase polymerase chain reaction (RT-PCR) method to study cytokine expression in astrocytes in response to exogenous agents and mechanical injury. By quantitative RT-PCR, it has been recently shown that TNFα/ interleukin (IL)-1β induced IL-6 in mouse results in astrocyte cultures. IL-6 belongs to a family of neuroactive cytokines including leukemia inhibitor factor (LIF) and ciliary neurotrophic factor (CNTF). RT-PCR has been used to demonstrate that the cholinergic differentiation factor/leukemia inhibitory factor (CDFLIF) and CNTF induce mRNAs for choline acetyltransferase, somatostatin, substance P, vasoactive intestinal peptide, cholecystokinin, and enkephalin in cultured sympathetic neurons. These data suggest that CDFLIF and CNTF may share receptor subunits and signal transduction pathways. Lipopolysaccharide, PMA, tumor necrosis factor (TNFα), and IL-lβ have also been found to induce LIF in cultured mouse astrocytes but not in the immortalized microglial cell line (BV-2) provided by Bocchini.

Inhibition of GFAP Synthesis with Antisense Nucleic Acid Constructs

Glial fibrillary acidic protein (GFAP), the major component of the intermediate filament in differentiated astrocytes (Eng et al., 1971; Eng, 1985), is extensively synthesized within and adjacent to the site of injury (Eng, 1988a; Condorelli et al., 1990; Hozumi et al., 1990; Vijayan et al., 1990). Other than GFAP accumulation, astrogliosis is also characterized by astrocyte proliferation (hyperplasia) and extensive hypertrophy of the cell body, nucleus as well as cytoplasmic processes (Eng, 1988a). Astrogliosis may participate in the healing phase following CNS injury by actively monitoring and controlling the molecular and ionic contents of the extracellular space of the CNS. They can wall off areas of the CNS that are exposed to non-CNS tissue environments following trauma. On the other hand, such responses may interfere with the function of residual neuronal circuits, by preventing remyelination, or by inhibiting axonal regeneration (Eng et al., 1987; Stensaas et al., 1987; Reier and Houle, 1988). Although astrogliosis has received considerable attention in term of its proposed inhibitory effect on CNS repair, there is still very little specific information available concerning the properties of reactive astrocytes, what triggers glial reactivity, and many of the cellular dynamics associated with scar formation. Control of astrocyte proliferation, differentiation, and astrogliosis may be linked to GFAP synthesis. Our aim was to transfect astrocytes with exogenous synthetic oligo- or polynucleotides, which would allow the manipulation of a transient suppression of GFAP synthesis which might delay the gliotic reaction and the scar formation, thus allowing neurons and oligodendrocytes to re-establish a functional environment.