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PubMed1995

Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia.

The expression of chemotactic receptors in the central nervous system is largely unexplored. In this study, we examined human astrocytes and microglia as well as the conditionally immortalized human astrocyte cell line HSC2 for expression of the C5a-anaphylatoxin receptor (C5aR), the interleukin-8 receptor (IL-8R) and the f-Met-Leu-Phe receptor (FMLPR). Using flow cytometry, indirect immunofluorescence and RT-PCR analysis, we demonstrated that astrocytes, microglia and HSC2 cells contain specific RNA and express surface protein for all three chemotactic receptors. These are the first studies to demonstrate definitively the expression of these chemotactic receptors astrocytes and microglia, thereby expanding the types of cells known to express chemotactic receptors. Moreover, these data suggest that these chemotactic receptors may play an important role in mediating the inflammatory response and perhaps other yet undescribed biological phenomena in the central nervous system.

Adult↗
PubMed1996

Apoptosis of cultured microglia by the deprivation of macrophage colony-stimulating factor.

Promotion of microglial proliferation and differentiation by colony-stimulating factors (CSFs) and disappearances of microglia at the late neonatal stage by decreasing of CSFs have been reported. In this study, the effects of the deprivation of macrophage CSF (M-CSF) on enriched microglia in cultures were examined by cytochemical methods including in situ nick-end labeling for DNA fragmentation, and Carrazi's hematoxylin nuclear staining. When M-CSF was deprived from the culture medium: (1) at least 40% of the cells were weakly labeled by nick-end within 3 h and more than 70% of the cells were clearly labeled by 16 h; and (2) nuclear condensation or fragmentation, and formation of apoptotic bodies were observed within 48 h. LeY-positive immunoreactivity, identified as a characteristic of cells undergoing apoptosis, was observed on cells positively labeled by nick-end and condensed nucleus, and ones budding apoptotic bodies. From these results, it is conceivable that microglia undergo apoptosis when M-CSF is deprived from the culture medium and, therefore, require CSFs for their survival. This in vitro phenomenon suggests that one of the mechanisms of microglial disappearance in vivo after synaptogenesis may be due to apoptosis by decreasing level of CSFs.

Animals↗
PubMed1996

Up-regulation of cystatin C by microglia in the rat facial nucleus following axotomy.

Cystatin C, a cysteine proteinase inhibitor, is expressed in the central nervous system (CNS) as well as many other organs of mammals. However, little is known concerning whether its expression is regulated under pathological conditions of the CNS and what types of cells are responsible for this regulation. We performed differential hybridization screening of cDNA libraries derived from the rat facial nucleus and found a cDNA of rat cystatin C to be up-regulated following facial nerve axotomy. In situ hybridization using an RNA probe for rat cystatin C revealed that cystatin C mRNA in the facial nucleus was markedly increased in amount by day 7 after axotomy and was then decreased to the normal level by day 50. The intense signal for cystatin C mRNA in the damaged facial nucleus was localized in the glial cells which had the morphological characteristics of microglia. Light and electron microscopic immunohistochemistry using a rabbit antibody specific for cystatin C confirmed that microglia in the damaged facial nucleus were strongly positive for cystatin C. The immunoreactivity was also found in the extracellular space, consistent with the fact that cells producing cystatin C generally secrete this protein. These results demonstrate that cystatin C is markedly up-regulated by microglia in response to axotomy and is probably secreted by these cells into the extracellular space, suggesting that this proteinase inhibitor has (a) significant function(s) in the processes of neuronal degeneration, regeneration, and/or repair subsequent to axotomy.

Animals↗
PubMed1995

Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain.

Senile plaques found in the brains of patients with Alzheimer's disease (AD) are surrounded by clusters of reactive microglia. Isolated human microglia placed in contact with plaques in vitro are activated to release a factor which is toxic to neurons. This same neurotoxin is found in AD brain tissue and causes damage to pyramidal neurons in vivo when infused into rat hippocampus. Highest concentrations of the neurotoxin are in those brain structures most burdened by reactive microglia, suggesting that plaque-activated cells contribute to the neuronal damage and impaired cognition seen in patients with Alzheimer's dementia.

Alzheimer Disease↗
PubMed1993

Expression of an outwardly rectifying K+ channel in rat microglia cultivated on teflon.

Membrane currents of cultured human monocytes and rat microglia were recorded with the whole-cell patch clamp technique. Freshly isolated monocytes or resting (proliferating) microglia express only inwardly rectifying K+ channels. However, incubation in teflon bags leads to the expression of additional, outwardly rectifying K+ channels. The outward K+ conductance of microglial cells was inhibited by intracellular Cs+ and extracellular 4-aminopyridine or tetraethylammonium. Functional similarities with the microglial outwardly rectifying K+ channel were found in the Kn-channel of lymphocytes which has recently been cloned (RGK5). The polymerase chain reaction (PCR) was used to demonstrate the presence of RGK5-like mRNA in microglia.

Animals↗
PubMed1993

Microglia express the type 2 plasminogen activator inhibitor in the brain of control subjects and patients with Alzheimer's disease.

Localization of type 2 plasminogen activator inhibitor (PAI-2) was investigated immunohistochemically in postmortem brain tissue of control subjects and patients with Alzheimer's disease (AD). Both the mouse monoclonal and the goat polyclonal antibodies to PAI-2 stained microglia. Reactive microglia were stained more intensely than resting microglia. PAI-2 may regulate the plasminogen activators and plasmin system in lesions of AD. It is suggested that PAI-2 plays a role for cell migration and matrix breakdown as well as for aberrant neurite outgrowth in senile plaques where persistent microglial activation is observed.

Aged↗
PubMed1994

Glutamate production by cultured microglia: differences between rat and mouse, enhancement by lipopolysaccharide and lack effect of HIV coat protein gp120 and depolarizing agents.

Glutamate release from rat and mouse microglia subcultures grown in a serum-free medium was substantially greater in the presence than in the absence of a physiological concentration of glutamine (0.5 mM). Mouse microglia produced and released more glutamate than rat microglia. Glutamate accumulation in the medium increased with time and cell density, which is consistent with the virtual absence of glutamate reuptake. Lipopolysaccharide (LPS; 10-100 ng/ml), HIV coat protein gp120 (0.1-10 nM), high K+ (35 mM) or ATP (150 microM), did not affect glutamate release from cells maintained in serum-free medium. In the presence of 1% dialyzed serum, however, LPS induced a dose- and time-dependent increase in the accumulation of glutamate in the medium, suggesting that, as in other cell types, serum factors are required for LPS binding to its receptors.

Adenosine Triphosphate↗
PubMed1995

Activated microglia cause superoxide-mediated release of iron from ferritin.

Ferritin contains the greatest part of the iron found in the brain, and the release of iron stores from ferritin has an essential role in iron-dependent lipid peroxidation. We examined the effect of cultured microglia on iron mobilization from ferritin. Microglia stimulated by phorbol myristate acetate caused the release of iron from ferritin, which was detected by monitoring iron-ferrozine complex formation. This iron mobilization was mediated by microglial superoxide production, as evidenced by the significant inhibitory effect of superoxide dismutase. The role of superoxide was also supported by the close correspondence of cumulative microglial superoxide production, as demonstrated by the MCLA (Cypridina luciferin analogue)-dependent chemiluminescence assay, to the time course of iron release from ferritin. Iron release induced by activated microglia may be partly responsible for the oxidative damage that is thought to occur in Parkinson's disease and other neurodegenerative disorders.

Animals↗
PubMed1995

Investigation of tau-2 positive microglia-like cells in the subcortical nuclei of human neurodegenerative disorders.

An anti-tau monoclonal antibody tau-2 was demonstrated to react with the cells which characteristically appeared in the subcortical nuclei of certain neurodegenerative disorders. These cells had rod-like cell bodies and elongated processes, whose morphology was consistent with that of reactive microglia (tau-2 positive microglia-like cells; TPMC). TPMC were diffusely scattered in the subcortical nuclei, especially the putamen, irrelevant to focal tissue injury such as infarcts and amyloid deposits. TPMC were positively immunostained with anti-ferritin antibody, but negatively with LN3, anti-GFAP, other kinds of anti-tau and anti-neurofilament antibodies. TPMC were found in some cases of Alzheimer type dementia and diffuse Lewy body disease, but not in the cases of Parkinson's disease, Pick's disease and control without neurological disorder. Similar microglia-like cells were found around infarctic foci and amyloid cores of senile plaques, regardless of the disorder. They were, however, different from TPMC in that they were positively immunostained with LN3.

Aged↗
PubMed1996

Effects of phorbol ester on intracellular Ca2+ and membrane currents in cultured human microglia.

The effects of protein kinase C (PKC) activation by phorbol ester on intracellular Ca2+ concentration ([Ca2+]i) and membrane currents in human microglia grown in culture were investigated. Treatment of microglia with phorbol myristate acetate (PMA) resulted in a large increase in [Ca2+]i in cells loaded with fura-2. The increased levels of [Ca2+]i were not altered following removal of the phorbol ester. In Ca(2+)-free medium, application of PMA did not increase [Ca2+]i. In addition, PMA application in standard Ca(2+)-solution containing lanthanum (1.8 mM) had no effect on the microglial response to PMA, suggesting that the phorbol ester actions were due to transmembrane influx of Ca2+ but not through voltage-gated Ca2+ channels. Whole-cell patch clamp measurements demonstrated that PMA potentiated an outward K+ current and inhibited an inward rectifier K+ current. This study is the first demonstration that PKC activation by phorbol ester leads to increased intracellular [Ca2+] and changes in membrane currents in human microglia.

Brain↗
PubMed1995

Cytokine-dependent K+ channel profile of microglia at immunologically defined functional states.

Microglia were enriched in brain cell cultures from newborn mice as a result of supplementation with the growth factors macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor. When separately administered these two cytokines promote the outgrowth of loosely adherent cells with similar morphology which stained positive for CD11b and nonspecific esterase. Microglial cells isolated from both types of culture were electrophysiologically characterized using the whole cell configuration of the patch-clamp technique. Different resting membrane potentials were measured. In response to hyperpolarizing and depolarizing voltage commands 68 of 91 macrophage colony-stimulating factor-cultured microglial cells exhibited only an inward rectifying potassium current. By contrast, an outward potassium current was observed on 71 of 95 granulocyte/macrophage colony-stimulating factor-grown cells. Parallel testing of their capability for antigen presentation proved the activated functional state of these microglial cells. They induce antigen-specific T cell response without prior stimulus. In comparison, cells developed with macrophage colony-stimulating factor failed to present antigen. In such resting microglia a short-term treatment with granulocyte/macrophage colony-stimulating factor or interferon-gamma provoked a strong appearance of outward potassium currents, however, only the interferon-gamma-trigger resulted in efficient antigen presentation. The differential induction of both functional parameters suggests the detection of outward potassium currents to provide an electrophysiological activation marker of microglia which is subjected to cytokine regulation but not compellingly linked to antigen presentation.

Animals↗
PubMed1996

Cyclin D1 messenger RNA is induced in microglia rather than neurons following transient forebrain ischaemia.

Following 30 min of forebrain ischaemia in the rat, delayed neuronal death occurs in the CA1 sector of the hippocampus within two to three days, whereas neurons in other selectively vulnerable regions, such as the dorsolateral striatum, die within 6-12 h. In this study, we investigated cyclin D1 expression, which codes for a regulatory protein in cell cycle regulation, but it is also induced in sympathetic neurons undergoing programmed cell death. Cyclin D1 messenger RNA could not be detected by in situ hybridization techniques in brains of control rats, but was found at one and two days after ischaemia in regions of the dorsolateral striatum with neuronal degeneration. DNA fragmentation in this region, identified by the terminal transferase biotinylated-UTP nick end labelling (TUNEL) procedure, was observed from 6 h after ischaemia onward. In the hippocampus, increased levels of cyclin D1 messenger RNA were found at two and three days after ischaemia in the striatum pyramidale of the CA1 sector. This expression was associated with the occurrence of neuronal damage and TUNEL-stained neurons. By seven days cyclin D1 messenger RNA was found in hardly any brain structure. There was no temporospatial overlap of cyclin D1 expression with the expression of the immediate-early genes c-fos, c-jun, and mkp-1, a result which is clearly distinct from findings in sympathetic ganglion neurons undergoing programmed cell death. These results do not suggest a role for cyclin D1 in neuronal cell death following transient forebrain ischaemia. The similarity of the cyclin D1 expression profile with that of the microglia-specific CR3 complement receptor beta-subunit messenger RNA, and the results of combined in situ hybridization and microglia-specific immunohistochemistry suggest that microglia are the source of cyclin D1 messenger RNA in the postischaemic brain. Since cyclin D1 codes for a critical regulatory protein for progression of the G0 to G1 phase in the cell cycle and we did not observe prominent occurrence of DNA fragmentation in microglial cells in the hippocampus at time points when cyclin D1 messenger RNA was found, we suggest that cyclin D1 induction is involved in the onset of microglial cell proliferation.

Animals↗
PubMed1996

Complement factor C5a and epidermal growth factor trigger the activation of outward potassium currents in cultured murine microglia.

Microglia, the resident macrophages of the brain, are transformed from a quiescent into an activated phenotype in a number of pathological conditions. The signalling mechanisms which control such transformations are not yet understood. In the present study, we have characterized fast electrophysiological responses in cultured microglia, induced by two putative signalling substances, complement 5a (C5a), a chemotactic agent for macrophages and microglia, and epidermal growth factor, the receptor of which is up-regulated during pathological conditions in the brain. Both factors transiently activate an outwardly rectifying K+ conductance, while the membrane of the unstimulated microglial cell is dominated by an inwardly rectifying K+ conductance. The C5a-stimulated current developed within about 20s and decayed within a slightly slower time course. It was activated by depolarlizing voltage steps positive to the resting membrane potential of about -70 mV, and neither inactivated nor showed a delayed activation following voltage steps. The epidermal growth factor-stimulated current showed similar characteristics. When G-proteins were specifically blocked, the K+ conductance could no longer be activated by C5a or epidermal growth factor, suggesting that for both agonists an inhibitory G-protein is involved in the intracellular signalling cascade. We tested if the induction of the K+ conductance is causally linked to other C5a-induced cellular responses, like transient cytosolic Ca2+ elevation and mobility. The K+ conductance was not activated when a Ca2+ transient was induced by thapsigargin, nor did a blockade of the C5a-induced K+ conductance by K+ channel blockers affect the motility response. This implies that after activation of the C5a receptor and the G-protein, the K+ conductance activation, the Ca2+ mobilization and the motility response are governed by independent intracellular pathways, and that the K+ conductance increase must serve other functions than the control of motility.

Animals↗
PubMed1995

Differential regulation by beta-amyloid peptides of intracellular free Ca2+ concentration in cultured rat microglia.

We have previously shown that exposure to beta-amyloid peptides alters microglial activity and viability. It is thought that beta-amyloid peptides induce toxicity in neuronal cultures by destabilizing Ca2+ homeostasis. To investigate the effects of beta-amyloid peptides on intracellular free Ca2+ concentration ([Ca2+]i) in cultured microglia, we used Fura-2 imaging. Exposure to 25 microM beta-amyloid-(25-35) induced increases in 2+]i within 1 h. In contrast, exposure to 25 microM beta-amyloid-(1-42), the full-length homolog to the beta-amyloid protein deposited in plaques, does not, over the same time period. However, the average [Ca2+]i of microglia is increased by a 6 h exposure to beta-amyloid-(1-42). Thus, beta-amyloid-(25-35) can alter [Ca2+]i in microglia on a different time scale than beta-amyloid-(1-42), indicating a specificity in the response of these cells as compared to neurons.

Amyloid beta-Peptides↗
PubMed2006

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine.

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.

Analysis of Variance↗
PubMed2004

Transglutaminase 2 induces nitric oxide synthesis in BV-2 microglia.

A hallmark of brain inflammation is the activation of microglia. Excessive production of nitric oxide (NO), as a consequence of increased inducible nitric oxide synthase (iNOS) in glia, contributes to neurodegeneration. Transglutaminase 2 (TGase 2) is a cross-linking enzyme, which is increased in neurodegeneration. TGase 2 is also considered to be a useful and reliable marker for activation levels in resident and inflammatory macrophages. Therefore, an increase of TGase 2 expression may contribute to activation of microglia. To test this hypothesis, we analyzed the expression of TGase 2 in BV-2 microglia activated with lipopolysaccharide (LPS). Total TGase activity was increased about 5-fold after 24h exposure to LPS. The increase of NO synthesis is correlated with increase of TGase 2 expression. Secretion of NO was reduced between 40 and 80% by TGase inhibition in a dose-dependent manner. This suggests that TGase 2 appears to control iNOS transcription.

Animals↗
PubMed2005

Effect of rottlerin, a PKC-delta inhibitor, on TLR-4-dependent activation of murine microglia.

In microglia, Toll-like receptors have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. The effect of rottlerin, a PKC-delta specific inhibitor, on TLR-4-mediated signaling was investigated in murine microglia stimulated with lipopolysaccharide and taxol. Pretreatment of microglia cells with rottlerin decreased LPS- and taxol-induced nitric oxide production in a concentration-dependent manner (IC50 = 99.1+/-1.5 nM). Through MTT and FACS analysis, we found that the inhibition effect of rottlerin was not due to microglial cell death. Rottlerin pretreatment also attenuated LPS-induced phosphorylation of IkappaB-alpha, nuclear translocation of NF-kappaB, and expression of type II nitric oxide synthase. In addition, microglial phagocytosis in response to TLR-4 activation was diminished in which rottlerin was pretreated. Together, these data raise the possibility that certain PKC-delta specific inhibitors can modulate TLR-4-derived signaling and inflammatory target gene expression, and can alter susceptibility to microbial infection and chronic inflammatory diseases in central nervous system.

Acetophenones↗
PubMed2006

Oxidized low density lipoprotein suppresses lipopolysaccharide-induced inflammatory responses in microglia: oxidative stress acts through control of inflammation.

Low density lipoprotein (LDL) is readily oxidized under certain conditions, resulting in the formation of oxidized LDL (oxLDL). Despite numerous in vitro reports that reveal the pathogenic role of oxidative stress, anti-oxidative strategies have underperformed in the clinic. In this study, we examine the role of oxLDL in brain inflammatory responses using cultured rat brain microglia. We demonstrate that oxLDL inhibits lipopolysaccharide (LPS)-induced inflammatory responses in these cells. It also decreases LPS-induced expression of inducible nitric oxide synthase and production of nitric oxide, and reduces LPS-induced secretion of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Oxysterols, known components of oxLDL and endogenous agonists of liver X receptor, can simulate the inhibitory effects of oxLDL in LPS-activated microglia. In addition, their inhibitory effects were mimicked by liver X receptor (LXR) agonists and potentiated by a retinoid X receptor agonist, suggesting these molecules heterodimerize to function as oxysterol receptors. Taken together, our results demonstrate that oxLDL inhibits LPS-induced inflammatory responses in brain microglia and that these inhibitory effects are mediated by oxysterols and, at least in part, by the nuclear receptor LXR. Our results suggest an additional mechanism of action for oxidative stress that acts indirectly via modulation of inflammatory responses. Although further studies are needed, these results answer in part the question of why anti-oxidative strategies have not been successful in clinical situations. Moreover, as brain inflammation participates in the initiation and progression of several neurodegenerative disorders, the present data provide information that should prove a useful guide for designing therapeutic strategies to combat oxidative brain diseases.

Animals↗