Primary astroglial cultures

Conclusion Primary cultures from neonatal rat brain consist mainly of astroglial cells, immunohistochemically identified by GFAp and S-100. As other cells than astrocytes may survice in the culture, specific markers for the expected cells were used. Cells with phagocytic properties, endothelial-like cells, oligoblasts, ependymal cells and mesenchymal cells were identified. No neurons have so far been detected.The astroglial cells have a high-affinity uptake for glutamate, aspartate GABA, taurine and hypotaurine, while there is probably a non-saturable uptake of norepinephrine, dopamine and 5-HT. The enzymes MAO, COMT, GABA-T and GS have been demonstrated. It thus seems that astrocytes take part in the inactivation of neurotransmitters, although amino acids and monoamines are taken up with different mechanisms.The presence of receptors for different neurotransmitters and neuromodulators has been demonstrated on astrocytes.Astroglial-enriched cultures from various brain regions have shown that the cells express specialized functional properties concerning neurotransmitter uptake, metabolizing enzymes and receptor density.Astroglial cell differentiation in culture is shortly reviewed and one possibility to affect this maturation by co-cultivation with neuronal containing cultures is point out.     

Characterization of insulin-like growth factor binding proteins of cultured rat astroglial and neuronal cells

Insulin-like growth factor binding proteins produced by cultured rat neurons, astrocytes, and rat cell lines BRL-3A and B104 were compared to binding proteins found in rat serum, using affinity labeling, deglycosylation, and Western ligand blotting studies. Each source elaborated an unique pattern of heterogeneous binding proteins. Some of the binding proteins from different sources behaved similarly in each experimental system suggesting that subsets of these binding proteins may be structurally related. In particular, our data suggest that cultured astrocytes and neurons make the major binding protein produced by BRL-3A cells.     

Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes. © 1996 Wiley-Liss, Inc.     

Neuronal inhibition of astroglial cell proliferation is membrane mediated

Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fluorescence microscopy or with [35S]methionine-labeled cells indicated that the kinetics of the binding of the neurons to astroglial cells are rapid, occurring within 10 min of the addition of the neurons to the growing glia. 6 h after neuronal attachment, astroglial DNA synthesis decreases, as shown by a two- to fivefold decrease in [3H]thymidine incorporation, and glial growth ceases. No effects on astroglial cell growth were seen after adding medium conditioned by purified cerebellar neurons cultured in the absence of astroglia, by astroglia cultured in the absence of neurons, or by a mixed population of cerebellar cells. This result was unchanged when any of these media were concentrated up to 50-fold, or when neurons and astroglia were cultured in separate chambers with confluent medium. Two groups of experiments suggest that membrane-membrane interactions between granule neurons and astroglia control astroglial cell growth. First, neurons fixed with dilute amounts of paraformaldehyde (0.5%) bound to the astroglia with the same kinetics as did living cells, inhibited DNA synthesis, and arrested glial growth within hours. Second, a cell membrane preparation of highly purified granule neurons also bound rapidly to the glia, decreased [3H]thymidine incorporation two- to fivefold and inhibited astroglial cell growth. The rate of the decrease in glial growth depended on the concentration of the granule neural membrane preparation added. A similar membrane preparation from purified cerebellar astroglial cells, PC12 cells, 3T3 mouse fibroblasts, or PTK rat epithelial cells did not decrease astroglial cell growth rates. Living neurons were the only preparation that both inhibited glial DNA synthesis and induced the astroglial cells to transform from the flat, epithelial shapes they have when they are cultured without neurons to highly differentiated forms that resemble Bergmann glia or astrocytes seen in vivo. These results suggest that membrane-membrane interactions between neurons and astroglia inhibit astroglial proliferation in vitro, and raise the possibility that membrane elements involved in glial growth regulation include neuron-glial interaction molecules.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Serological characterization of macrophage hybridomas: identification of an interferon-gamma-inducible surface marker

Macrophage hybridoma clones prepared by fusion of splenic adherent cells with P388D1 tumor cells have previously been shown to be heterogeneous with respect to function at the clonal level. In this study the macrophage clones were phenotypically characterized by indirect RIA using a battery of rat MAbs to murine myeloid and lymphoid cell surface markers. All macrophage clones expressed the common leukocyte antigen T200 and the Mac-1 alpha and beta chains. Markers which were differentially expressed among the clones included class II antigens and the antigens detected by MAbs MIV 55, MIV 38, and 14G8. The antigens detected by the latter three MAbs were referred to as MBR-1, -2 and -3, respectively. Functional heterogeneity did not correlate with phenotypic heterogeneity among the macrophage clones. Treatment of macrophage clones with IFN-gamma resulted in a significant increase in the expression of class II antigens and induced the expression of MBR antigens on some clones which were constitutively negative for these markers. The clonal distribution and induction patterns of class II antigen as compared to MBR antigen indicated that regulation of expression of these markers was independent. In addition, the clonal distribution and induction pattern of MBR antigens, along with competitive binding studies using radiolabeled MIV 38 and 14G8 MAbs, suggested that the three MBR antigens were similar or closely associated molecules.     

Monoclonal antibodies to Streptococcus mutans serotype f polysaccharide cross-react with the 'mutans group' of oral streptococci

Abstract Eleven stable clones producing monoclonal antibodies (MAbs) against serotype polysaccharide f were generated from rat IR 983F myeloma cells and splenocytes from rats immunized with formalin-killed whole Streptococcus mutants OMZ 175. These MAbs were IgG 2b, they had different affinities towards polysaccharide f and all but three were able to inhibit saliva binding to S. mutants OMZ 175 cells. Two specificities were recognized on S. mutans serotype f polysaccharide. One MAb (K24) recognized an epitope on the poly-rhamnose core, as determined by competitive ELISA using different sugars. The ten other MAbs recognized an epitope which seems principally formed of glucose subunits. Purified polysaccharides from other 'mutans streptococci' were able, to different extents, to inhibit the binding of the MAbs to polysaccharide f, confirming the presence of common epitopes in the different strains.     

Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides.

We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGB19, GMR2, GMR7, GGR12, GMR5, and GGR13 reacted strongly with the gangliosides GD3, O-Ac-GD3, GD2, GD1b, GT1b, and GQ1b, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-Ac-GD3, GQ1b, and GT1a, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GD1b, GT1b, and GQ1b on mouse, rat, and human leukemia cells. GD1b was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GT1b nor GQ1b was detected in these cell lines.     

Binding of antigen in the absence of histocompatibility proteins by arsonate-reactive T-cell clones

Inducer T-cell clones reactive to the p-azobenzenearsonate (arsonate) hapten possess binding sites for radioactive arsanylated proteins, which are not present on clones with other antigen specificities. Binding occurred in the absence of histocompatibility proteins. Binding was specific for the p-azobenzenearsonate hapten, since unconjugated proteins and proteins conjugated to the nonactivating o-azobenzenearsonate hapten neither bound to the clones nor competed binding of radioactive antigen. One of the clones was studied in more detail, using a panel of structural analogs of arsonate conjugated to the carrier protein ovalbumin. All conjugates that activated the clone in the presence of antigen-presenting cells also competed binding of radioactive antigen in the absence of antigen-presenting cells. Nonactivating conjugates did not compete binding. Based on evidence in this and the succeeding paper (Rao et al., accompanying paper), we suggest that these arsonate-binding sites may include the physiological antigen receptors of arsonate-reactive T-cell clones.     

The effect of Ndrg2 expression on astroglial activation

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2′-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology.     

人抗E．Coli J5噬菌体抗体制备的初步研究

以E．Coli J5株对合人Ig基因的噬菌体抗体库进行淘筛富集,免疫印迹筛选,以及ELISA检测,结果获得4株能与E．Coli J5株结合的阳性克隆,且阳性克隆结合抗原的活性可分别被E．Coli J5株、E．Coli Rc-LPS和抗E．Coli J5株核心糖脂域MAb抑制．PCR检测表明,4株阳性克隆均分别带有约660bp大小的重链和轻链基因片段．SDS－PAGE与蛋白质印迹的结果显示,经IPTG诱导的阳性克隆能表达分子量约为50000大小的蛋白,提示该4株阳性克隆能够表达具有一定抗原结合活性的人源Fab片段．     

Insulin effect on GABA uptake in astroglial primary cultures

Astroglial cultures from newborn mouse cerebral cortex contain [¹²⁵I]insulin binding sites. Binding was specific reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [¹²⁵I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time-and dose-dependent inhibition of the [³H]GABA high affinity uptake. A decrease in theV _max rather than, an effect on theK _m was observed. This effect was dose-dependent and effective at 10^–10 M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [³H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin of [³H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate, that insulin may interfere in neuromodulation through astroglial signals.     

Long-term effects of brain trypsinization before cell seeding on cell morphology and surface composition

The relation between the pattern of proteins localized in the surface of astroglial cells and cell differentiation was investigated in primary cultures derived from neonatal rat brains, dissociated either mechanically (MDC) or by 3 (TDC3) and 30 minutes (TDC30) trypsinization. Morphological and ultrastructural studies revealed a bed layer composed of flat, polygonal young and differentiated astrocytes in all types of cultures and a surface layer composed of small, ovoide undifferentiated cells which were more numerous in TDC30 than in TDC3 and MDC. The enrichment in undifferentiated cells, induced by prolonged brain trypsinization prior cell seeding, was observed during two weeks in culture; latter, by day 20, the cell population in all cultures was that of differentiated astrocytes. The presence of structural and enzymatic cell markers indicated that the cell population in MDC and TDC3 as well as in TDC3, including the small cells, was of astroglial origin. Concomitant with the morphological changes, cells in TDC30 were less accessible to surface labeling than those composing MDC. Subsequent electrophoresis of the labeled surface proteins demonstrated that a 140-130 K complex was the most "sensible" to brain trypsinization and that their accessibility to the surface probing was maximal during the differentiation of astrocytes in MDC or of small cells in TDC30. By day 20, these components were not significantly labeled in both, MDC, and TDC30, cultures. The use of two types of astrocytes primary culture which were different in the ratio of differentiated to undifferentiated cells and their surface labeling at different growth stages showed a variation in the composition of surface proteins during the cell maturation. The increased accessibility of some surface proteins to external probing when the cells developed to differentiated astrocytes might suggest their involvement in cell differentiation.     

Comparative analysis of the membrane proteins and their specificities in neurons,protoplasmic astrocytes,and oligodendrocytes from rat brain

Plasma membranes from neuronal perikarya (N), protoplasmic astrocytes (A) and oligodendrocyies (O) of rat brains were analysed with respect to their protein and glycoprotein contents and specificities. SDS-polyacrylamide gel electrophoresis revealed a total number of 23, 17, and 17 major proteins in N, A, and O respectively. Periodate-Schiff's staining showed that approximately 40–60% of these proteins were glycoproteins. The reactivity of these glycoproteins to Con A and WGA was also studied. Selective iodination of whole cells followed by electrophoresis and autoradiography indicated that of the major proteins, only 25% of neuronal and 60% of astroglial and oligodendroglial membrane proteins were exposed outside the cell surface. The overall results suggest that membrane proteins of each of the three cell types studied here have characteristically different internal and external markers differing in size, glycoprotein content, and reactivity of the glycoproteins to lectins.     

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.     

Utilization of mannose by astroglial cells

Uptake and metabolism of mannose were studied in astroglia-rich primary cultures derived from neonatal rat brains. A saturable component of mannose uptake was found with half-maximal uptake at 6.7±1.0 mM mannose. In addition, a non-saturable component dominated the uptake at high concentrations of mannose. Glucose, cytochalasin B, or phloretin in the incubation buffer inhibited the carrier-mediated uptake of mannose. Within the astroglial cells mannose is phosphorylated to mannose-6-phosphate. In cell homogenates, the K_M value of mannose-phosphorylating activity was determined to be 24±7 M. The V_max value of this activity is only 40% that of glucose-phosphorylating activity. Mannose-6-phosphate was converted to fructose-6-phosphate by mannose-6-phosphate isomerase. The specific activity of this enzyme in homogenates of astroglial cultures was higher than that of hexokinase. Two products of mannose utilization in astroglial cells are glycogen and lactate. The amounts of each of these products increased with increasing concentrations of mannose. In contrast to the generation of lactate, that of glycogen from mannose was enhanced in the presence of insulin. In conclusion, we suggest that mannose is taken up into the cells of astroglia-rich primary cultures by the glial glucose transporter and is metabolized to fructose-6-phosphate within the astroglial cells.     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.