An in vitro model for the study of microglia-induced neurodegeneration: involvement of nitric oxide and tumor necrosis factor-alpha.

The precise function of activated microglia and their secretory products remains controversial. In order to assess the role of microglial secretion products, we established an in vitro model of an inflammatory reaction in the brain by co-culturing microglial and neuronal cell lines. Upon stimulation with interferon-gamma and lipopolysaccharides, the microglial cells adopted an activated phenotype and secreted tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2) and nitric oxide (NO). Neuronal degeneration was quantified by measuring the concentrations of microtubule associated protein tau and neuron specific enolase, which are also used as diagnostic tool in Alzheimer's disease, in supernatants. In activated contact co-cultures, the levels of these neuronal markers were significantly raised compared to non-activated co-cultures. NO-synthase inhibitors significantly diminished the rise of tau in activated co-cultures, while indomethacin, superoxide dismutase, or a neutralizing TNF-alpha antibody did not. When a chemical NO-donor or TNF-alpha were added to pure neuronal cultures, cell viability was significantly reduced. TNF-alpha increased neuronal sensitivity towards NO. There were indications that a part of the cells died by apoptosis. This model demonstrates a neurotoxic role for NO in microglia-induced neurodegeneration and provides a valuable in vitro tool for the study of microglia-neuron interactions during inflammation in the brain.

Interleukin-4 enhances the in vitro precursor cell recruitment for tumor-specific T lymphocytes in patients with glioblastoma.

Previously the authors showed that interleukin-4 (IL-4), used in combination with IL-2, increases the reduced proliferation rate of T cells of glioblastoma-bearing patients after in vitro autologous immunization. In this report, they sought to determine whether this effect is caused by a direct mitogenic effect of IL-4, or rather by an indirect effect through an increased expression of the IL-2 receptor subunits or an enhanced recruitment of responsive cells. Flow cytometric analysis confirmed that the IL-2 receptor subunits are less expressed on circulating T cells from patients with glioblastoma than on those from healthy donors. Because no significant modification of the expression of the p55 and p75 subunits of the IL-2 receptor is observed in cultures treated with both IL-2 and IL-4, the reported enhanced proliferation rate cannot be attributed to an increased level of IL-2 receptor expression. Limiting dilution assays, using autologous target cell immunization, show that treatment with both cytokines (IL-2 plus IL-4) significantly increases the number of recruitable precursor cells without affecting their proliferation rate. These results indicate that IL-4 facilitates an immune response against the autologous tumor cells in glioblastoma-bearing patients by increasing the recruitable precursor T-cell frequency.

Stimulation of endothelin B receptor modulates the inflammatory activation of rat astrocytes.

Inside the brain tissue, endothelins play numerous important biological roles. One of the targets, astrocytes, predominantly display endothelin receptor subtype B (ET(B)). On cultured primary rat astroglial cells, we analyzed the effect of IRL1620, a selective ET(B) receptor agonist, on the production of nitric oxide (NO) and the synthesis of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. We performed these experiments in the presence or absence of interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). IRL1620 decreases NO production under basal conditions and after IFN-gamma stimulation. However, during LPS-induced NO production, IRL1620 enhances this release. The basal IL-6 secretion and especially the LPS-induced synthesis are enhanced by the IRL1620 stimulation. The LPS-dependent TNF-alpha production is increased by the ET(B) stimulation. The IRL1620-induced decrease of basal NO production is not dependent on Ca2+ entry or on phospholipase C (PLC) activation, as shown by the use of LaCl3 and U73122, respectively. In the presence of LPS, the IRL1620 potentiation of NO production is inhibited by LaCl3 and U73122. The IRL1620-induced increase of IL-6 is dependent on PLC activation. These results suggest that endothelins can have dual effects depending on the costimulatory factors present. Endothelins thus have important immunomodulatory functions in the brain.

Regional heterogeneity of the astroglial immunoreactive phenotype: effect of lipopolysaccharide.

In order to find out whether the concept of regional heterogeneity in astrocytes also applies to the immunoreactive phenotype, we studied cultured primary rat astrocytes originating from five different brain regions (cortex, hippocampus, striatum, septum, and brainstem).We investigated this heterogeneity through the ability of lipopolysaccharide (LPS) to differentially induce several parameters that are known to characterize activated astroglia: major histocompatibility complex (MHC) class II and intercellular adhesion molecule (ICAM)-1 expression, nitric oxide (NO) production, synthesis of interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). Under basal conditions, some of these parameters are already heterogeneic. The presence of LPS enhances these differences: expression of MHC class II increases after a 48-hour incubation with LPS, with brainstem and hippocampus astrocytes reaching the highest levels; NO production is induced by an LPS incubation, with the brainstem showing low NO production levels; septum and striatum instead show higher cytokine (TNF-alpha, IL-6) productions. The baseline expression of ICAM-1 also shows major regional differences, with the brainstem displaying the highest ICAM-1 expression. Our results demonstrate that the immunoreactive abilities of astrocytes show regional heterogeneities. This specialization may be implicated in the pathophysiological pathways of several neurodegenerative disorders.

Cultured astrocytes express regional heterogeneity of the immunoreactive phenotype under basal conditions and after gamma-IFN induction.

Cerebral astrocytes are known to show a region-specific phenotype, concerning the expression of several receptors and the synthesis of secreted substances. In order to find out whether this heterogeneity also exists for the immunological activation, we studied several parameters that are known to characterize activated astroglia on cultured primary rat astrocytes originating from cortex, hippocampus, striatum, septum and brain stem: major histocompatibility complex (MHC) class II and intercellular adhesion molecule (ICAM)-1 expression, nitric oxide (NO) production and interleukin-6 (IL-6) synthesis. Unstimulated cultures show a baseline expression of MHC class II molecules that differs from one region to another, hippocampus and brain stem showing the highest values. These differences are strongly enhanced after a 48-h incubation with gamma-interferon (gamma-IFN). NO production is also induced by a 72-h incubation with gamma-IFN, showing similar patterns of regional specialization. The baseline expressions of ICAM-1 and IL-6 also show major regional differences, with the brain stem and the striatum showing elevated values for ICAM-1, and the septum and the brain stem producing the largest amounts of IL-6. The expressions of ICAM-1 and IL-6 are not affected by an incubation with gamma-IFN. Our results demonstrate that the immunological activities of astroglial cells show regional heterogeneities. This specialization may be implicated in the pathophysiological pathways of several neurodegenerative disorders.

Human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5 respond to fMLP with [Ca2+]i variation and O2- production.

In the presence of interleukin-3 and interleukin-5, eosinophil precursors from human umbilical cord blood mononuclear cells were regularly differentiated into mature eosinophil-like cells expressing normal morphology and cyanide-resistant peroxidase. O2- production and [Ca2+]i rise were measured in these in vitro differentiated eosinophils after fMLP stimulation; with dihydrorhodamine-123 and fura-2, respectively. Umbilical cord blood-derived eosinophils responded to fMLP (0.01 nM to 3 microM) with a concentration-dependent production of O2- (EC50 = 63.1 +/- 17.2 nM; Emax = 71.0 +/- 6.2 pmol/min/10(6) cells). O2- production was correlated with an fMLP concentration-dependent increase in [Ca2+]i (EC50 = 32.5 +/- 14.9 nM; Emax = 200.0 +/- 23.9 nM). These results indicate that human umbilical cord blood-derived eosinophils demonstrate functional characteristics similar to adult human peripheral blood eosinophils after activation by fMLP. Therefore, the large numbers of eosinophils (2-3 x 10(6)/ml cord blood) which can be obtained by culture of human cord blood mononuclear cells may serve as a useful model for future studies which will provide insight into the pathogenesis of diseases associated with eosinophils.

Interleukin-4 down-regulates MHC class II antigens on cultured rat astrocytes.

Mammalian cerebral astrocytes can be brought to express major histocompatibility complex (MHC) class II molecules upon appropriate stimulation. It is well established that this expression is subject to modulation by several neurotransmitters and cytokines. We show that the low, basal expression of MHC class II antigens on cultured rat astrocytes is concentration-dependently down-regulated by low concentrations of interleukin-4 (IL-4), reaching maximal inhibition at 10 U/ml. The higher, gamma-IFN-induced, expression of class II molecules is also decreased by increasing concentrations of IL-4, significant effects being already observed at 5 U/ml. Since the cAMP as well as the nitric oxide dependent cGMP pathway have previously been shown to mediate an inhibition on astroglial MHC class II expression, we measured the intra-cellular content of cyclic nucleotides after stimulation with IL-4. No rise in cAMP or cGMP is detected. Similarly, IL-4 does not affect the induced synthesis of nitric oxide radicals. Since MHC class II expression is a critical step in many regulatory processes of the cellular immune reaction, IL-4, via its activity on astroglial cells, emerges as an important modulator of immunological activities in the central nervous system.

Nitric oxide modulates gamma-interferon-induced MHC class II antigen expression on rat astrocytes.

Brain astroglial cells can be brought in vivo and in vitro to express an immunocompetent cell-like phenotype. We investigated the effect of the NO. releasing compound sodium nitroprusside (SNP) on Ia expression in rat astrocyte cultures. SNP down-regulates, in a concentration-dependent manner, the gamma-interferon-induced Ia expression. Inhibition of the NO. synthesis attenuates the glutamate mediated down-regulation of class II expression. Our results show that NO. is implicated in the immunomodulatory reactions in the brain parenchyma.

Interleukin-4 affects phenotype and proliferative response of peripheral blood lymphocytes from glioblastoma patients after specific or nonspecific in vitro stimulation.

Immunosuppressive events are often observed in glioblastoma-bearing patients. We tested the response of circulating lymphocytes from glioblastoma patients to low concentrations of interleukin (IL)-4 and IL-2 after lectin activation or specific in vitro stimulation by autologous tumor cells. In the presence of IL-2, IL-4 up-regulates the proliferation rate of phytohemagglutinin (PHA)-P-stimulated glioblastoma patients' peripheral blood lymphocytes (PBL)s. Allogeneically- and syngeneically-stimulated PBLs of these patients present an increased proliferation rate in the presence of IL-4. This specifically stimulated lymphocyte population presents a very low proportion of CD8+ cells. This proportion is slightly increased in the presence of IL-4. Our results indicate that the glioblastoma cell-imposed inhibition on T-cells can be partly overcome by low concentrations of IL-4 during in vitro stimulation. Our experiments also demonstrate that glioblastoma-bearing patients' PBLs constitute a good model in which to study the effects of IL-4.

Suloctidil increases the rat brain cortex microvascular regeneration after a lesion.

A "cavity" lesion made by aspiration in the rat occipital cortex induces a parenchymal and a vascular reaction in its vicinity. The first was mainly characterized by cellular necrosis and gliosis, the second by an increase of the vascular network. In vehicle treated rats, a 50% significant increase of the vascular network was observed around the cavity 4 days after the lesion, in comparison to the uninjured contralateral cortex. The effects of a vasoactive substance, suloctidil, on the vascular reaction was studied in the brain cortex. A single oral dose of suloctidil (30 mg/kg; 2 hours before the sacrifice) gave the same effect as the vehicle group. After 8 days of suloctidil oral administration (30 mg/kg; twice daily: 4 days before lesion and 4 days after) a significant increase (123%) of the vascular network was observed around the cavity. The hypothetical ways by which a chronic treatment of suloctidil induces this increase of the neovascularization observed after cortical lesion are discussed.

Topographical distribution in the adult rat brain of neurotrophic activities directed to central nervous system targets.

Cortex, hippocampus, septum and striatum of day 18 rat embryos were grafted to several brain regions of young adult rats which had been lesioned in the chosen area 4 days earlier. Thirty days after transplantation, the grafts were fixed and morphometrically analysed under light microscope. The volumes, neuronal densities and total number of neurons of the transplants were compared. Each graft survived best when transplanted to its original region. Good survival was also achieved by heterotopic grafts between regions that are anatomically related. Striatal grafts showed reasonable survival only when transplanted to their original site. In a second series of experiences, the neurons from the same embryonic brain regions were cultured in a defined medium, to which was added tissue extracts from the lesioned regions of the adult brain. The neuronal survival was estimated. The in vitro results are closely related to those obtained in vivo. This experimental evidence agrees with the theory of the existence of a retrograde transport of NGF from the hippocampus to the septum, sustaining the survival of the latter. On the other hand, our results demonstrate the existence of other unidentified neurotrophic factors in the central nervous system which differ from one region to another.

A new model for quantification of microvascular regeneration after a lesion of the rat cerebral cortex.

The purpose of this study is to validate a method for the quantification of the neovascularization in the vicinity of a lesion made in the cerebral rat cortex. A cavity, made by aspiration in the occipital cortex of young rats, induces around the lesion a parenchymal and vascular reaction. The parenchymal reaction is characterized by cellular necrosis and gliosis. The vascularization is more dense around the cavity than in normal cortex. Morphometric analysis indicates, 8 days after the lesion, a 130% increase of the total length of the vessels in comparison to the contralateral normal cortex.

The abilities of the rat brain to sustain the survival of implanted or cultured embryonic dorsal root ganglion neurons depend on the selected region.

The purpose of the present study was to examine the regional distribution of trophic activity in the lesioned adult rat brain on implanted peripheral neurons as target cells. Embryonic dorsal root ganglia (DRG) were implanted into selected regions of previously lesioned adult rat brains. The survival of these neurons was quantified after thirty days. In another experiment embryonic DRG neurons were cultured for 24 h together with tissue extracts of the same lesioned brain regions and the neuronal survival was estimated. We report here that all the tested regions, i.e. the occipital, parietal and frontal cortex, the hippocampus and the striatum, exert a trophic effect on embryonic DRG neurons both 'in vivo' and 'in vitro'. This effect is twice as high in the hippocampus as in the striatum, the other cortical regions showing intermediate values. The in vitro results are qualitatively the same as the in vivo results.