Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

T-cell costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are expressed in human microglia but not in astrocytes in culture

The B7-1 and B7-2 expressed on the 'professional' antigen-presenting cells (APC) of the lymphoid system are counterreceptors for the T cell antigens CD28/CTLA-4. The B7/CD28 interaction provides a critical costimulatory signal in the decision between functional activation or clonal anergy of T cells. To investigate the biological role of B7 in the central nervous system, constitutive and cytokine-induced expression of B7 was investigated in fetal human astrocytes and microglia in culture. B7-1 expression was minimally detectable in unstimulated microglia but was increased markedly following exposure to IFN-γ or GM-CSF. B7-2 was expressed at a high level in untreated microglia and upregulated to a small degree by exposure to IFN-γ or GM-CSF. In contrast, B7-1 and B7-2 were undetectable in astrocytes under unstimulated or IFN-γ/GM-CSF-treated conditions. These results indicate that both B7-1 and B7-2 are expressed in cultured human microglia but not in astrocytes.     

Reduction of Iba1-expressing microglial process density in the hippocampus following electroconvulsive shock

Recent studies place emphasis on the modulations of immune system in various psychiatric disorders and/or treatments. The aim of this study was to investigate the implications of immune-related glial cells in a rapid-acting treatment for depression, namely, electroconvulsive therapy (ECT). Specifically, the effects of electroconvulsive shock (ECS; animal model of ECT) on microglia were morphologically determined in the mouse hippocampus by using ionized calcium-binding adaptor molecule 1 (Iba1) immunocytochemistry. For comparison, S100β-positive astrocytes, another type of glial cells, were also tested. After 24 hours of acute ECS administration, a meshwork of Iba1-positive microglial processes was largely diminished, although the change was transient. In mice that received chronic ECS administration, the decline of Iba1-positive microglial process meshwork continued even 1 month after the last shock. Morphometric image analysis revealed the significant reduction of Iba1-positive microglial process density following ECS administration. On the other hand, neither acute nor chronic ECS administration made alterations in the patterns of expression of S100β immunoreactivity. No significant changes were detected in the cell surface area of S100β-positive astrocytes following ECS administration. The optical disector analysis demonstrated that ECS did not affect the numerical densities of Iba1-positive microglia and S100β-positive astrocytes in the hippocampus. These results provide some key to understand the potential role of microglia and astrocytes in the antidepressant action of ECT.     

Interferon-γ Induces Apoptosis and Augments the Expression of Fas and Fas Ligand by Microglia in Vitro

Activation of microglia by interferon-γ (IFN-γ) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-γ has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-γ-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-γ (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-γ concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-γ also potentiated the expression of Fas and FasL in a similar dose–response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-γ may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-γ modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes.     

An alpha5beta1 integrin inhibitor attenuates glioma growth

Integrins are heterodimeric transmembrane proteins, which mediate cell–cell and cell–extracellular matrix (ECM) interaction. We show, that an inhibitor of alpha5 beta1 integrin (α5β1), JSM6427, attenuated glioma growth and decreased the density of microglia at the tumor border. 21 days after glioma cell injection into an experimental mouse model, the tumor volume was significantly smaller after treating animals for 14 days with JSM6427 as compared to controls. We could demonstrate the expression of integrin α5β1 on both microglia and glioma cells using flow cytometry. In a slice culture we could compare glioma growth in the presence and absence of microglia. Slices injected with glioma cells were treated with the integrin inhibitor JSM6427 and showed a significant reduction in tumor size as compared to control. Depleting microglial cells from the slice culture by treatment with clodronate liposomes abrogated the effect of JSM6427 on glioma invasion indicating that the presence of microglia is required. We show further, that microglial migration, and proliferation was attenuated dose-dependently by JSM6427.     

Activation of nuclear factor-κB by β-amyloid peptides and interferon-γ in murine microglia

An increasing body of evidence suggests that amyloid-β (Aβ) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of Aβ towards microglia, we investigated the ability of Aβ peptides to activate nuclear factor (NF)-κB in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of Aβ(25–35) and 100 U/ml IFNγ resulted in the detection of a specific NF-κB DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-κB complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-κB/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with Aβ(25–35) plus IFNγ. Higher concentrations of Aβ(25–35) were effective by themselves in inducing NF-κB activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-κB inducer. As expected, LPS strongly induced the formation of two NF-κB DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-κB activation might constitute one of the mechanisms underlying the inducible expression of κB-dependent genes in microglia stimulated by Aβ peptides and IFNγ, or by LPS.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.     

Integrin Mac-1 and β-amyloid in microglial release of nitric oxide

The β-amyloid protein associated with Alzheimer's disease (AD) has been well characterized biochemically; however, its primary biological function and mode of action in AD has not been determined. We have shown previously that β-amyloid (β25–35), in combination with interferon-γ (IFN-γ), can induce nitric oxide release from cultured hippocampal microglial cells. In the present study, binding of β-amyloid with the leukocyte integrin Mac-1, a cell surface receptor on microglia, was studied by observing (1) inhibition of β-amyloid (β25–35)-mediated release of nitric oxide from cultured microglial cells following exposure to monoclonal antibodies against Mac-1 (anti-CD18 and anti-CD11b) and (2) competitive binding of fluorochrome-labeled β25–35 with anti-CD18 or anti-CD11b using fluorescent flow cytometry. Wt.3 (anti-CD18 antibody) and OX42 (anti-CD11b antibody) were as effective as opsonized zymosan at inducing the release of nitric oxide from microglia. Furthermore, Wt.3 and OX42 acted synergistically to induce maximum nitric oxide release. An interaction between β-amyloid and CD18 of Mac-1 was evidenced by the suppressive action of β25–35 on Wt.3-mediated release of nitric oxide and the synergistic action between OX42 and β25–35 in inducing nitric oxide release from microglia. The tissue culture study was supported by competitive binding assays of fluorochrome-labeled β25–35 and Mac-1 antibodies (Wt.3 or OX42). The majority of microglial cells (71%) did bind biotinylated β-amyloid in the presence of cytochalasin B, suggesting that β-amyloid binding to microglia is a receptor-mediated event. Furthermore, pre-exposure to Wt.3, but not OX42, significantly decreased binding of biotinylated β25–35 to microglia. These findings suggest that CD18 of Mac-1 may play a role in β-amyloid-mediated release of nitric oxide.     

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ_1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ_1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Cytokine induction of heat shock protein expression in human oligodendrocytes: An interleukin-1-mediated mechanism

In this study, we examined the role of cytokines, known to be in elevated levels in multiple sclerosis (MS) plaques, in regulating oligodendrocyte (ODC) expression of heat shock protein (hsp) in human brain-derived glial cell cultures. Using dual-stain immunohistochemistry, we initially compared the ability of a mixture of cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-8, TNF-α, TNF-β, IFN-β and IFN-γ) with that of physical stimuli such as heat shock and peroxide, to increase cellular expression of the mainly inducible hsp72 species in mixed glial cell cultures (containing ODC, astrocytes and microglia). Similar to heat shock and peroxide, the cytokine mixture induced hsp72 expression only in ODC (70 ± 5% vs. a baseline of 3 ± 1% positive cells). When used individually, however, only IL-1α (79 ± 3%), IFN-γ (70 ± 2%) and TNF-α (65 ± 5%) induced ODC hsp72 expression in mixed glial cell cultures. In purified ODC preparations, only IL-1α induced hsp72 expression (84 ± 4%). An IL-1 receptor antagonist (IL-1ra), abrogated hsp72 induction by IL-1α (16 ± 3%) as well as that due to IFN-γ (14 ± 1%) and TNF-α (13 ± 2%) in mixed glial cell cultures. Furthermore, ODC express IL-1 receptors, detected by confocal laser scanning microscopy. Our data indicate that cytokines mediate hsp induction in ODC possibly via a final common pathway involving IL-1 binding to its receptor on ODC. Such interaction could enhance any putative ODC-immune interactions which are dependent on hsp molecule recognition.     

Production of interleukin-12 and expression of its receptors by murine microglia

Production of interleukin-12 (IL-12) by cultured murine microglia and astrocytes was examined, by means of ELISA to detect heterodimeric p70 and RT-PCR to analyze the expression of mRNA encoding p35 and p40. Microglia, but not astrocytes, produced IL-12 p70 in response to lipopolysaccharide and interferon-γ. The microglial cell line, Ra2, produced only p40, but not p35, upon above stimulation. Thus, it is possible that some population of microglia induce helper 1 type T cell response via producing IL-12 in the CNS. Microglia were induced to express mRNA encoding IL-12 receptors which were exclusively expressed in activated T and NK cells.     

Amyloid-β peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-β (Aβ) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Aβ 1–42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1β. Aβ also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Aβ 1–42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Aβ 1–42 sequence or Aβ 17–42 had little or no effect. We also determined that the Aβ activity can be found in a supernatant fraction containing soluble Aβ oligomers. Our data suggest that Aβ plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.     

Differential suppression of interferon-γ-induced Ia antigen expression on cultured rat astroglia and microglia by second messengers

The roles of intracellular second messengers in interferon-γ (IFN-γ)-induced Ia antigen (Ag) expression by astroglia and microglia were examined. Ia Ag on both glia types was induced by IFN-γ. Reagents known to increase intracellular cAMP or activate intracellular protein kinase C (PKC) reduced IFN-γ-induced Ia Ag expression by astroglia. In contrast, increasing intracellular cAMP had no suppressive effect on Ia Ag expression by microglia. These results indicate (1) cAMP and PKC negatively regulate IFN-γ-induced Ia expression on astroglia, and (2) Ia expression is regulated differentially in astroglia vs. microglia. These findings may explain the frequent observation of Ia⁺ microglia (or macrophages) but not astroglia in various neurodegenerative diseases.     

Cultured Rat Microglia Express C1q and Receptor for C1q: Implications for Amyloid Effects on Microglia

Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process-bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds β1-42, the major constituent of senile plaques, and since β1-42 is toxic to microglia in vitro , we asked if preincubation of β1-42 with C1q alters either metabolic indices of amyloid-induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that preincubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to β1-42 on β1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-γ. While these data exclude a role for the C1q receptor in β1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-γ would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques.     

Transforming growth factor β's 1, 2 and 3 inhibit proliferation of ramified microglia on an astrocyte monolayer

The transforming growth factor β's (TGFβ) are a multipotent family of cytokines with strong immunosuppressive and neurotrophic effects. In the current study, we examined the effect of the TGFβ's 1, 2 and 3 on the proliferation of ramified microglia cultured on top of a confluent astrocyte monolayer. All three TGFβ isoforms inhibited proliferation. PCR analysis also showed the presence of mRNA for the TGFβ receptors type I and II and for all 3 TGFβ isoforms in microglia, astrocytes and in co-cultures. Moreover, removal of this endogenous TGFβ activity with antibodies against TGFβ1 and TGFβ3 strongly stimulated microglial proliferation. These inhibitory effects on the proliferation of ramified microglia suggest that TGFβ's may play an important role in the regulation of the microglial population under normal conditions and after injury or disease in the central nervous system.