Detailed analysis of inflammatory and neuromodulatory cytokine secretion from human NT2 astrocytes using multiplex bead array

Astrocytes are a very important cell type in the brain fulfilling roles in both neuroimmunology and neurotransmission. We have conducted the most comprehensive analysis of secreted cytokines conducted to date (astrocytes of any source) to determine whether astrocytes derived from the human Ntera2 (NT2) cell-line are a good model of human primary astrocytes. We have compared the secretion of cytokines from NT2 astrocytes with those produced in astrocyte enriched human brain cultures and additional cytokines implicated in brain injury or known to be expressed in the human brain. The concentration of cytokines was measured in astrocyte conditioned media using multiplex bead array (MBA), where 18 cytokines were measured simultaneously. Resting NT2 astrocytes produced low levels (～1-30 pg/ml) of MIP1α, IL-6 and GM-CSF and higher levels of MCP-1, IP-10 and IL-8 (1-11 ng/ml) under non-inflammatory conditions. All of these in addition to IL-1β, TNFα, and IL-13, were increased by pro-inflammatory activation (TNFα or IL-1β stimulation). In contrast, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, LTα, and IFNγ were not detected in astrocyte conditioned media under any of the culture conditions tested. NT2 astrocytes were unresponsive to IL-2 and the adenyl cyclase agonist, forskolin. Interestingly, IFNγ stimulation selectively increased IP-10 secretion only. As astrocytes stimulated with IL-1β or TNFα produced several chemokines in the ng/ml range, we next assessed the chemoattractant properties of these cells. Conditioned media from TNFα-stimulated astrocytes significantly chemoattracted leukocytes from human blood. This study provides the most comprehensive analysis of cytokine production by human astrocytes thus far, and shows that NT2 astrocytes are highly responsive to pro-inflammatory mediators including TNFα and IL-1β, producing cytokines and chemokines capable of attracting leukocytes from human blood. We conclude that in the absence of adult human primary astrocytes that NT2-astrocytes may provide a valuable alternative to study the immunological behaviour of human astrocytes.     

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Effect of Ro 5-4864 and PK11195 on protein phosphorylation in mitochondria isolated from primary cultures of rat astrocytes

Astrocytes are the most numerous cell type within the central nervous system. Earlier, high-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of astrocytes. TSPO/PBR was shown to be localized in mitochondria. Recently, we showed that TSPO/PBR ligands, Ro 5-4864 and PK11195, were able to modulate the function of non-specific pore (PTP) in brain and liver mitochondria as well as protein phosphorylation in the presence of threshold calcium concentrations. In the present study for the first time the function of astrocyte mitochondria were studied under condition of PTP opening. Parameters of PTP induction were measured by means of simultaneous registrations of the membrane potential, calcium accumulation and calcium release as well as detection of the oxygen consumption with selective electrodes. Four phosphorylated proteins in range of 67 kDa, 46 kDa, 48 kDa and 3.5 kDa have been found under these conditions. It was established that in astrocyte mitochondria TSPO/PBR exists in monomer form (18 kDa). The phosphorylation level of these proteins was found to be modulated by TSPO/PBR ligands, Ro 5-4864 and PK11195, in a range of concentrations from 0.01 to 1 μM, in the same way as it was earlier described for brain mitochondria [Azarashvili et al., J Neurochem., 2005].     

Specific binding of benzodiazepines to human breast cancer cell lines

Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.     

The Peripheral-Type Benzodiazepine Receptor Is Present in Astrocytes but Is Not a Primary Site of Action for Convulsants/Anticonvulsants

Abstract: High-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of both mouse neocortical and cerebellar astrocytes. The binding sites were enriched in mitochondrial fractions on differential centrifugation. An 18-kDa polypeptide was specifically photolabelled in cerebellar astrocytes by [³H]-PK 14105, a photolabel for the peripheral-type benzodiazepine receptor. However, this polypeptide did not show any reactivity with an antiserum previously raised against the corresponding polypeptide from rat adrenal gland. Various anticonvulsant and convulsant agents were tested for their ability and potency at inhibiting [³H]Ro 5-4864 binding to neocortical astrocytes. Many of these compounds, previously reported to be inhibitors of diazepam binding to neocortical astrocytes, proved ineffective in this study. No correlation was observed between convulsant/anticonvulsant potency and ability to inhibit [³H]Ro 5-4864 binding to the peripheral-type benzodiazepine receptor in these cells. Thus, whereas some convulsants and anticonvulsants might interact with this astrocytic receptor, such a system has no validity as a general screening method for these agents.     

Transient acidification and subsequent proinflammatory cytokine stimulation of astrocytes induce distinct activation phenotypes

The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukocyte entry. Cultured astrocytes typically have a polygonal morphology until stimulated. We hypothesized that cultured astrocytes which were induced to stellate would have an activated phenotype compared with polygonal cells. We investigated the activation of astrocytes derived from adult macaques to the cytokine TNF‐α under resting and stellated conditions by four parameters: morphology, intermediate filament expression, adhesion, and cytokine secretion. Astrocytes were stellated following transient acidification; resulting in increased expression of GFAP and vimentin. Stellation was accompanied by decreased adhesion that could be recovered with proinflammatory cytokine treatment. Surprisingly, there was decreased secretion of proinflammatory cytokines by stellated astrocytes compared with polygonal cells. These results suggest that astrocytes are capable of multiple phenotypes depending on the stimulus and the order stimuli are applied. J. Cell. Physiol. 228: 1284–1294, 2013. © 2012 Wiley Periodicals, Inc.     

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.     

Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes.

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.     

Behavioural actions of Ro 5-4864: A peripheral-type benzodiazepine?

Ro 5-4864 is a 1,4 benzodiazepine lacking typical benzodiazepine behavioural actions, and which has very low affinity for the “classical” CNS benzodiazepine binding sites. However, Ro 5-4864 has very high affinity for the peripheral type of binding site in the periphery and in the brain. Evidence is reviewed that Ro 5-4864 is sedative, convulsant and anxiogenic in rodents. We also describe the effects of combining Ro 5-4864 treatment with benzodiazepines (e.g. diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). The binding sites that might be mediating these behavioural actions of Ro 5-4864 are discussed.     

Cytoskeletal Alterations in Human Fetal Astrocytes Induced by Interleukin-1β

Abstract: Previous studies in this and other laboratories have shown that interleukin-1β (IL-1β) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL-1β (rhIL-1β) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL-1β, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein-phalloidin binding. These “activated” astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL-1β stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL-1β-stimulated astrocytes revealed a transient, marked decrease in steady-state levels of mRNA for GFAP, vimentin, and microtubule-associated protein 4. The decrease in mRNA levels was evident by 4–8 h and fell to the lowest level at 16–24 h (80–98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL-1β stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL-1β for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL-1β-stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an “activated” state of astrocytes.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Evidence that the Peripheral-Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β-Endorphin Release from AtT-20 Cells by Blockade of Voltage-Dependent Calcium Channels

The demonstrations that Ro 5-4864, a ligand selective for the peripheral-type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral-type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5-4864 disrupted a common cellular regulatory event. Using a well-characterized anterior pituitary-derived tumor cell line (AtT-20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), beta-lipotropin hormone (beta-LPH), and beta-endorphin (BE), we have investigated the molecular mechanism of action of Ro 5-4864's capacity to alter BE secretion. Ro 5-4864 inhibits basal and induced BE release from AtT-20 cells, through a cyclic AMP-independent mechanism. Ro 5-4864 completely blocked the corticotropin-releasing hormone and forskolin-induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT-20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate voltage-dependent Ca2+ channels, resulted in a cyclic AMP-independent, dose-related increase in BE secretion. This CGP-induced BE release was blocked by increasing concentrations of Ro 5-4864. In contrast to the capacity of Ro 5-4864 to block CGP-induced BE release, Ro 5-4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage-dependent Ca2+ channels. Our findings suggest that Ro 5-4864 inhibits BE secretion from AtT-20 cells through a blockade of the voltage-dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5-4864's diverse effects observed on other cell types.     

Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor

The effect of cytokines on astrocytes cultured from mature bovine brain was determined both in a serum-containing medium and in a chemically-defined medium. The results showed that in serum-free medium, human TNF and, to a lesser degree, IL-6 and lymphotoxin, were mitogenic for astrocytes. Increased uptake of [3H]thymidine could be detected within 36 h in vitro and its presence in astrocytes was confirmed by autoradiography. In contrast, neither IL-1 alpha nor IL-1 beta induced astrocyte proliferation in serum-free medium but showed some synergistic effect with serum after 72 h. The proliferative effect of TNF and IL-6 was confirmed by cell counting. None of the cytokines tested was toxic for astrocytes as measured by 51Cr release. No mitogenic effect for oligodendroglia, purified from the same source, was detected. The results support a role for products of activated inflammatory cells in the development of astrocyte proliferation that may contribute to the reactive gliosis found in white matter diseases of the central nervous system such as multiple sclerosis.     

Microglia are the major source of TNF-α and TGF-β1 in postnatal glial cultures; regulation by cytokines,lipopolysaccharide, and vitronectin

Damage to the central nervous system (CNS) leads to increased production of TNF-α and TGF-β1 cytokines that have pro- or anti-inflammatory actions, respectively. To define whether astrocytes or microglia express these cytokines, prior studies have used mixed glial cultures (MGC) to represent astrocytes, thought these results are inevitably complicated by the presence of contaminating microglia within MGC. To clarify the cellular source of these cytokines, here we employed a recently described method of preparing microglia-free astrocyte cultures, in which neural stem cells (NSC) are differentiated into astrocytes. Using ELISA to quantify cytokine production in three types of glial culture: MGC, pure microglia or pure astrocytes, this showed that microglia but not astrocytes, produce TNF-α, and that this expression is increased by LPS, IFN-γ, and to a lesser extent by vitronectin, but decreased by TGF-β1. In contrast, TGF-β1 was produced by microglia and astrocytes, though at 10-fold higher levels by microglia. TGF-β1 expression in microglia was increased by vitronectin and to a lesser extent by TNF-α and LPS, but astrocyte TGF-β1 expression was not regulated by any factor tested. In summary, our data reveal that microglia, not astrocytes are the major source of TNF-α and TGF-β1 in postnatal glial cultures, and that microglial production of these antagonistic cytokines is tightly regulated by cytokines, LPS, and vitronectin.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Tumor necrosis factor alpha and interleukin-1 alpha inhibit through different pathways interferon-gamma-induced antigen presentation, processing and MHC class II surface expression on astrocytes, but not on microglia

Astrocytes and microglia, two glial cell populations of the CNS, have been described to be involved in many immune processes. We used defined combinations of cytokines, interferon gamma (IFN-gamma)/interleukin-1 alpha (IL-1 alpha) and IFN-gamma/tumor necrosis factor alpha (TNF alpha), to simulate different in vitro immune environments observed in disease or inflammation. In these conditions, we analyzed and compared the regulating effects of these cytokines on cell surface and total expression of MHC II and on the capacity of murine astrocytes and microglia to present peptide and native antigens to specific primed T cells. Neither IL-1 alpha nor TNF alpha affected the IFN-gamma-induced antigen presentation capacity of microglia. Astrocytes, however, were severely impaired in their capacity to present native antigens and, to a minor extent, a peptide antigen. Total expression of MHC II was not affected by these cytokines in microglia, whereas in astrocytes it was reduced by IL-1 alpha and increased by TNF alpha. Both cytokines downregulated MHC II expression at the surface of astrocytes, but not of microglia. This shows that TNF alpha affects the of IFN-gamma-immunocompetent astrocytes to process and present antigen, probably either by altering membrane traffic of MHC II and of antigen and/or enzymatic activities associated with these mechanisms, while IL-1 alpha does so by downregulating MHC II expression. Altogether, our results illustrate how differently astrocytes and microglia react toward a defined, similar immune environment. One type of cell, the astrocytes, downregulate their T-cell stimulation and MHC II trafficking, and probably also their antigen processing, functions while the other, the microglia, maintain their antigen presentation potential.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

Pharmacological and physiological properties of benzodiazepine binding sites in rodent brown adipose tissue

[3H]Diazepam and [3H] Ro5 -4864 were used as ligands to identify and characterize peripheral-type benzodiazepine binding sites in mouse and rat brown adipose tissue (BAT) membranes. [3H]Diazepam and [3H] Ro5 -4864 binding sites in BAT are pharmacologically similar to peripheral-type benzodiazepine binding sites in other tissues. Stimulators of central-type benzodiazepine receptors had no effect on or inhibited ligand binding to BAT membranes. Brown adipose tissue benzodiazepine binding sites are highly localized to mitochondria-containing subcellular fractions. These binding sites decrease with age in BAT from Fischer 344 rats. Stimulation of BAT thermogenesis in mice with 1-norepinephrine led to a decrease in [3H] Ro5 -4864 binding in the tissue.