Human Fetal Astrocytes Research Articles

Cytokine production by human fetal microglia and astrocytes. Differential induction by lipopolysaccharide and IL-1 beta.

As part of a study on the role of cytokines in central nervous system development and dysfunction, we determined the pattern of cytokine production in highly purified cultures of microglia and astrocytes isolated from second-trimester human fetal brains. Levels of TNF-alpha, IL-1 beta, and IL-6 mRNA and protein were determined by Northern blot analysis and ELISA before and after stimulation with LPS, TNF-alpha, or IL-1 beta. In microglia, LPS induced mRNA for all three cytokines. High protein levels of IL-6 and TNF-alpha were also found in the medium, whereas IL-1 beta protein was mostly cell associated. IL-1 beta also induced message for all three cytokines, in the rank order of IL-1 beta > IL-6 > TNF-alpha. TNF-alpha induced mRNA and protein for IL-1 beta but not for TNF-alpha or IL-6. In contrast, LPS failed to stimulate either mRNA or protein expression for any of the three cytokines in astrocytes. On the other hand, IL-1 beta provided a strong stimulus for astrocytes. IL-1 beta induced mRNA and protein for both TNF-alpha and IL-6, but the kinetics of the response differed for the two cytokines. TNF-alpha mRNA and protein levels peaked early (at 4 h and 16 h, respectively) and were undetectable by 72 h, whereas IL-6 mRNA peaked later (at 16 h) and protein levels continued to accumulate in the medium through 72 h. IL-1 beta did not induce IL-1 beta mRNA or protein in astrocytes. TNF-alpha did not induce expression of any of the cytokines in astrocytes. In conclusion, our results demonstrate that cytokine production can be induced in human fetal microglia and astrocytes but that the stimuli for induction differed significantly for the two cell types. Whereas LPS was a potent stimulus for microglia, astrocytes primarily responded to IL-1 beta. The data further suggest that microglia may be key regulators of astrocyte response, working primarily through the expression of cell-associated IL-1 beta.

Macrophage colony-stimulating factor in human fetal astrocytes and microglia. Differential regulation by cytokines and lipopolysaccharide, and modulation of class II MHC on microglia.

CSF-1 is a growth factor that selectively promotes the proliferation, survival, and differentiation of cells of the mononuclear phagocyte series. As part of a study on the role of cytokine and hematopoietic growth factors in central nervous system (CNS) development and inflammation, we examined the expression of CSF-1 in dissociated glial cells cultured from human fetal CNS tissue. CSF-1 mRNA and protein were constitutively expressed by astrocytes. The steady state level of CSF-1 mRNA was markedly up-regulated by both IL-1 beta and TNF-alpha in a time- and dose-dependent manner, whereas only a minimal increase was detected after stimulation with LPS. In unstimulated astrocyte cultures, CSF-1 protein levels gradually increased to 3.5-fold base-line values by 96 h and were significantly increased by all three stimulants in the order of IL-1 > or = TNF > LPS. Low levels of CSF-1 mRNA and protein were also detected in unstimulated microglia cultures. In contrast to astrocyte cultures, CSF-1 mRNA and protein increased significantly after stimulation with LPS, but changed only minimally after exposure to TNF-alpha or IL-1 beta. The effect of CSF-1 on cell proliferation, morphology, and class II MHC Ag expression was determined in highly enriched cultures of microglia and astrocytes. Microglia treated with CSF-1 showed a modest level of proliferation and differentiation into rod-shaped cells, whereas neither cell number nor shape was changed in astrocyte cultures. Interestingly, marked inhibition of both basal and IFN gamma-induced class II MHC Ag expression was observed in microglial cells cultured in the presence of CSF-1, whereas no effect was detected in astrocytes. These results suggest the possibility that in situ production of CSF-1 in the CNS may regulate normal glial cell development and contribute to the immunologic status of the CNS through the down-regulation of class II MHC expression.

Lymphocyte emperipolesis in human glial cells.

Astrocytes have been observed to contain intact, viable lymphocytes within their cytoplasm (emperipolesis) in multiple sclerosis plaques and some brain tumors. This study characterizes the adhesive, emperipoletic and phagocytic properties of glial cells in culture. Human fetal and adult astrocytes engaged in adhesion and emperipolesis of lymphocytes. Emperipolesis, and not adhesion, was temperature- and cation-dependent. The CD8 and MHC Class I antigens played a role in emperipolesis. Lymphocytes most often remained viable within the cytoplasm of astrocytes but occasionally underwent lysis or caused disruption of the astrocyte intermediate filaments. The phenomenon of emperipolesis is distinct from phagocytosis, since microglia showed prominent phagocytic properties but did not engage in emperipolesis. Conversely, astrocytes were efficient emperipolites and rarely demonstrated phagocytic properties.

Immunocompetent cell markers in human fetal astrocytes and neurons in culture.

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregulatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex-class II antigen HLA-DR was observed in the cytoplasm and on the cell surface of GFA-P+ astrocytes and increased with time in culture and cell passages. Among the T-lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4-6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B-lymphocyte marker CD21, the cellular receptor for Epstein-Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B-cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events.

Differential proliferative response of human and mouse astrocytes to gamma-interferon.

We have previously shown that gamma-interferon promoted the proliferation of adult human astrocytes isolated from brain biopsy specimens. In contrast, in the present study, astrocytes derived from neonatal mouse brains and treated with recombinant murine gamma-interferon responded by a decrease (average of 50% at 100 U/ml) in proliferation. The basal rate of proliferation as assessed by bromodeoxyuridine incorporation was markedly increased in neonatal mouse astrocytes when compared to the adult human cells, suggesting that age, and the corresponding metabolic activity of cells, could be important determinants in the mitogenic response of astrocytes to cytokines. However, subsequent examinations of fetal human and adult mouse astrocytes, with comparable basal rate of proliferation to neonatal mouse and adult human cells respectively, showed gamma-interferon to promote DNA synthesis in fetal human astrocytes while inhibiting that of adult mouse astrocytes. The results suggest species differences in the proliferative response of human and mouse astrocytes to the cytokine gamma-interferon.

Temperature-dependent modulation of lipopolysaccharide-induced interleukin-1 beta and tumor necrosis factor alpha expression in cultured human astroglial cells by dexamethasone and indomethacin.

In bacterial meningitis, LPS induces production in cerebrospinal fluid of the cytokines IL-1 beta and tumor necrosis factor alpha (TNF alpha), which are the principle mediators of meningeal inflammation. IL-1 beta and TNF alpha induce fever, and elevated temperature may affect cytokine expression. Dexamethasone treatment improves outcome in bacterial meningitis possibly by inhibiting IL-1 beta and TNF alpha. In this report, the effects of elevated temperature and dexamethasone on LPS-stimulated IL-1 beta and TNF alpha mRNA gene expression and protein synthesis were studied in human astrocytoma cell lines and primary cultures of human fetal astrocytes. Cells cultured at 40 degrees C exhibited smaller peaks of IL-1 beta and TNF alpha transcription and protein synthesis compared with cells cultured at 37 degrees C. The addition of dexamethasone before, during, or after exposure of the cells to LPS resulted in temperature-dependent inhibition of IL-1 beta transcription and protein synthesis. The most extensive inhibition occurred in pretreated cells cultured at 37 degrees C. Cotreatment with LPS and dexamethasone also inhibited TNF alpha mRNA transcription at both temperatures. The effects of another antiinflammatory agent, indomethacin, on LPS induction of IL-1 beta and TNF alpha mRNA were temperature and cell line dependent. These findings provide a possible explanation for the efficacy of dexamethasone treatment of bacterial meningitis and support the proposal that fever may be beneficial to the host in this disease.

Increased Susceptibility of Human Fetal Astrocytes to Human T-Lymphotropic Virus Type I in Culture

Human T-lymphotropic virus type I (HTLV-I) has been considered as an agent responsible for tropical spastic paraparesis and HTLV-I associated myelopathy. However, the pathogenesis of the diseases remains unclear. In a previous study we demonstrated that HTLV-I could infect adult human astrocytes and oligodendrocytes in vitro, although the rates of infected cells were low, at a rate of 0.1% and 0.01-0.05% respectively. Since mother-to-child transmission has been proposed as one of the major pathways for the prevalence of HTLV-I endemic, in the present study we investigated the susceptibility of human fetal astrocytes to HTLV-I in culture. After two days of co-culturing fetal brain cells with irradiated MT-2 cells (an HTLV-I-producing T-cell line), immunofluorescence staining revealed many positive astrocytes for HTLV-I p19 antigen. Multinucleated giant cells doubly immunoreactive to glial fibrillary acidic protein and HTLV-I antigen were frequently observed and showed a characteristic feature of hairy or fluffy external appearance. The percentage of infected astrocytes became as high as 19.4% at Day 21 of co-culture and then decreased. Electron microscopic examination revealed type C virus-like particles in astrocytes. These results indicate that human fetal astrocytes are more susceptible to HTLV-I infection than adult human astrocytes in tissue culture.

Stimulation of human fetal astrocyte proliferation by bacterial lipopolysaccharides and lipid A

This report concerns the effect of bacterial endotoxin [lipopolysaccharide(LPS) and lipid A] on cultured human fetal astrocytes. Exposure to 1 micrograms/ml LPS or lipid A caused a striking stimulation of the rate of proliferation of the cells. The effect was most pronounced with exponentially growing cultures. Stimulation was associated with enhance DNA synthesis as ascertained by [3H]thymidine incorporation. These findings at the cellular level may be of relevance in the elucidation of the effects of bacterial endotoxins on the developing human brain.

The induction of intercellular adhesion molecule 1 (ICAM-1) expression on human fetal astrocytes by interferon-λ, tumor necrosis factor α, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation

Antigen presentation reactions are dependent upon the expression of the class II major histocompatibility antigens (MHC), the T-cell receptor, and the presented antigen. Recent studies demonstrate that such processes also require the presence of adhesion molecules such as lymphocyte functional antigen 1 (LFA-1) and its cell surface ligand, intercellular adhesion molecule 1 (ICAM-1). It has been suggested that the brain astrocyte can function as a facultative antigen presenting cell (APC). This hypothesis is based upon th ability to induce the expression of the class II MHC antigens on astrocytes, and on their ability to present myelin basic protein on encephalitogenic T-cells in vitro. The best in vivo data showing that astrocytes serve as intracerebral APCs is the finding that astrocytes in multiple sclerosis plaques are DR + (class II MHC in human). However, it still remains to be resolved whether the in vivo expression of the MHC antigens in disease states is instrumental to antigen presentation mechanisms or whether these cell suface glycoproteins are expressed secondary to brain immune responses. If astrocytes function as immunocompetent APCs within the brain, it would seem that they would also be able to express molecules important for intercellular adhesion. Here, we present the first data that indicates that human astrocytes are capable of expressing ICAM-1 in response to cytokines that either induce or upregulate the expression of DR. In essence, cytokines derived from different cell types seem to exert similar pleiotropic effects on the modulation of MHC and ICAM-1 expression on astrocytes. This suggests that mechanisms critical to the initiation of brain immune responses involve a dynamic molecular interface between a variety of resident central nervous system and peripheral cell types.

Human Fetal Astrocytes in Culture Support the Growth of the Neurotropic Human Polyomavirus, JCV

JC virus (JCV) has frequently been described as a tissue specific agent in the human population targeting the myelin producing oligodendrocyte whose destruction results in the demyelinating disease, progressive multifocal leukoencephalopathy (PML). JC virus growth in cell culture systems has generally reflected that observation because its multiplication is most efficient in cultures derived from human fetal brain. These cultures, however, are composed of a mixed population of cells phenotypically categorized as either astrocytes or precursors to oligodendrocytes. We have been able to separate mixed populations of glial cells from human fetal brain in culture and produce a pure population of astrocytes. We then analyzed the astrocyte cell cultures for their ability to support JCV transcription, replication, and virion production. Our results demonstrate that cultures of astrocytic cell types support JCV gene expression leading to virus multiplication. These astrocytic cell cultures also can be passaged several times in culture without loss of their astrocytic phenotypes or susceptibility to JCV infection. These data are consistent with our studies of JCV gene expression in fixed brain tissues of PML patients and virus immortalized astrocytic cell lines. These results strongly suggest that astrocytes as well as oligodendrocytes play a role in the pathogenesis of PML and should focus additional experiments on this particular cell type.

DNA synthesis by young adult human-derived astrocytes in vitro

We have assessed whether adult human non-neoplastic astrocytes exhibit DNA synthesis in vitro, as measured using a double immunofluorescence technique to detect incorporation of 5-bromodeoxyuridine (BrdU) by nuclei of glial fibrillary acidic protein (GFAP)-containing cells. Dissociated cell cultures containing GFAP + cells were established from surgically resected temporal lobe tissue from 3 young adult individuals operated upon to remove epileptogenic foci. In 5–18-day-old cultures from each of the 3 individuals, we observed GFAP + cells whose nuclei had incorporated BrdU. BrdU nuclear staining was found in GFAP + cells with either flat or process-bearing morphologies. The mean mitotic index for the GFAP + cells was 6% (range 2–12%) as compared to a mean mitotic index of 29% for the GMK-7 glioma cell line. Our results do indicate that astrocytes derived from young adult donors, unlike such cells derived at autopsy from elderly adults, are capable of DNA synthesis in vitro, albeit to a markedly lesser extent than reported for fetal human astrocytes.

Detection of a nerve growth factor receptor on fetal human Schwann cells in culture: absence of the receptor on fetal human astrocytes

Cultures of human Schwann cells and astrocytes were established from fetal nerves and brains respectively. The human Schwann cells in culture expressed a nerve growth factor (NGF) receptor as determined by indirect immunofluorescence, autoradiography, and immunoprecipitation. In contrast, the human astrocytes in culture did not have an NGF receptor. Cultures of rat Schwann cells and astrocytes were also established for comparison, with similar results. The rat Schwann cells had an NGF receptor whereas the astrocytes did not. The functional significance of this NGF receptor on Schwann cells, as well as the lack of the receptor on astrocytes, is discussed.

Expression of receptors, linked to cyclic AMP formation by cultures of human fetal astrocytes.

Expression of receptors, linked to cyclic AMP formation by cultures of human fetal astrocytes.

Growth factors for fetal and adult human astrocytes in culture

Using a recently developed method for the determination of cell proliferation in culture, we assessed the effects of several growth factors on their capability to promote proliferation of astrocytes derived from human fetuses or adults. Factors tested included fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, glial growth factor from bovine pituitary, nerve growth factor, interleukin-2, dibutyryl cyclic adenosine monophosphate, and 4β-phorbol 12,13-dibytyrate. The results show that astrocytes from human adults do not undergo proliferation in vitro even in the presence of these agents and that in the case of fetal astrocytes, only glial growth factor from bovine pituitary, platelet-derived growth factor and the phorbol ester were mitogenic. In addition, the capability of fetal astrocytes to proliferate appears to decrease with the increasing length of time they have been maintained in culture.

Cultured human fetal astrocytes can be induced by interferon-γ to express HLA-DR

Interferon-γ (IFN-γ) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-γ. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-γ. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-γ treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, DRw52, DRw53 and DQw1. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-γ in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Effects of meso-2,3-dimercaptosuccinic acid on methylmercury-injured human fetal astrocytes in vitro

Monolayer cultures of human fetal astrocytes (HFA) were exposed to 0.01 m M methyl-mercuric chloride (MMC) for 30 to 60 min and were then treated with medium containing 1.0 m M meso-2,3-dimercaptosuccinic acid (DMSA), a newly synthesized complexing agent for heavy metals. Within 30 to 40 min of DMSA addition, the coagulated membrane of astrocytes injured by MMC recovered to a normal state with return of active filopodial and ruffling activity. Addition of DMSA prior to MMC exposure prevented the occurrence of degenerative changes in astrocytic membranes. Exposures to 1.0 m M DMSA alone for 24 hr did not cause degenerative changes in HFA. Thus DMSA appears to be an extremely effective agent in complexing methylmercury either in the membrane or in the culture medium with no demonstrable toxic actions on HFA in culture. The minute to minute sequential changes of HFA before and after exposures to MMC and DMSA were recorded with time-lapse cinematography and were repeated numerous times with similar results. Electron microscopic study revealed separation of membranes from cell bodies and processes associated with marked edema of the underlying matrix. The cytoskeletal elements associated with membranes became dissociated and disappeared after MMC exposure but promptly returned to normal with addition of DMSA and replacement with normal fresh culture medium.

Effects of methylmercury on human fetal neurons and astrocytes in vitro: A time-lapse cinematographic, phase and electron microscopic study

Time-lapse cinematography of organotypic cultures of human fetal cerebrum demon-strated cessation of neuronal migration following the addition of 0.02 m m methylmercuric chloride (MMC) to the culture medium. This was due to rapid cytotoxic effects of MMC characterized by cessation of filopodial activity, focal swelling, and partial fragmentation of neurites. Separation of the membrane from neurites associated with marked swelling of the matrix was prominent, especially at growth cones. Soon total fragmentation occurred at these sites resulting in separation of degenerated neurites from neuronal cell bodies in 1 to 4 hr. Addition of 0.1 m m MMC caused fragmentation and separation of neurites from most neuronal cell bodies in approximately 40 min while exposures to 0.2 m m MMC caused a similar phenomenon in approximately 20 min. The cytotoxic effect of MMC was stopped immediately and further degeneration did not occur when cultures exposed to MMC were washed and replaced with normal complete culture medium. When mercury was given in a highly polar sulfonic acid form ( p-chloromercuriphenyl sulfonic acid) the membrane of neurites and astrocytes remained intact, indicating that penetration of the membrane by mercury is necessary to initiate the toxic effects of MMC. At the ultrastructural level damage to plasma membranes and neurotubular injury are conspicuous early features of methylmercury toxicity, taking place more rapidly in neurons than astrocytes, and eventuating in irreversible degeneration of both cell types.

Effects of methylmercury on DNA synthesis of human fetal astrocytes: a radioautographic study

Methylmercury (MeHg), an organomercurial, is one of the most prevalent and toxic environmental pollutants. The major route of human exposure occurs via consumption of MeHg-adulterated seafood. Historically, MeHg epidemics in Minamata Bay, Japan, and in Iraq provided evidence of its developmental toxicity and neurological damage. This chapter describes MeHg kinetics in the human body and focuses predominantly on mechanisms involved in neuropathological and reproductive effects. The chapter also discusses the differential susceptibility to MeHg in fetuses and adults, with a special emphasis on its aberrant effects in the developing central nervous system. Discussions on risk assessment, treatment, and future directions are also covered in this chapter.

Effects of methylmercury on DNA synthesis of human fetal astrocytes: A radioautographic study

Effects of methylmercury on DNA synthesis of human fetal astrocytes: A radioautographic study

GLYCOSAMINOGLYCANS IN ASTROCYTES

By means of the Alcian blue ‘critical electrolyte concentration’ technique and specific enzymic extraction, stainable material corresponding to chondroitin sulphate has been demonstrated in the cytoplasm of reactive astrocytes around a needle wound in the brain of rats, in astrocytic processes in the white matter of the mouse brain after treatment with cuprizone, and in human fetal astrocytes grown in uitro. Increased amounts of glycosaminoglycans in astrocytes seem to accompany rearrangement of tissue components in the central nervous system, and the histochemical demonstration of chondroitin sulphate in astrocytes may reflect changes in the state of their cell surface.