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At least 703 records · Page 39Linked to original sources
PubMed2004

Tumor necrosis factor reduces cAMP production in rat microglia.

cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, mainly by regulating microglial activation and orienting these cells toward a neuroprotective phenotype. In order to elucidate the intracellular pathways regulated by tumor necrosis factor (TNF) in glial cells, I have studied the modulation of cAMP accumulation by TNF in microglia and astrocyte cultures obtained from the neonatal rat brain. Pre-treatment of microglia with TNF reduced in a dose- and time-dependent manner cAMP accumulation induced by forskolin (FSK), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). The TNF inhibitory action was 90% reverted by a neutralizing polyclonal anti-TNF antibody and was not prevented by a 16 h pre-treatment of microglial cultures with the Gi protein inhibitor pertussis toxin (PTx). These results suggest that TNF acts at a step of the cAMP transduction pathway other than receptors, G proteins, and phosphodiesterases. The target of TNF appeared to be adenylyl cyclase, whose ability to synthesize cAMP was markedly reduced (up to 50%) in membranes prepared from TNF-treated microglial cells, both in basal conditions and after stimulation with FSK. TNF induced a time-dependent degradation of IkappaB-alpha in microglial cells that was reverted by two inhibitors of nuclear factor kappaB activation, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-CBZ-Leu-Leu-Leu-al (MG132). The same inhibitors also markedly prevented the reduction of FSK-evoked cAMP accumulation by TNF, suggesting the involvement of NFkappaB in the regulation of adenylyl cyclase by TNF in microglia. Conversely, cAMP accumulation in astrocytes was not affected by TNF. Based on these findings, it is proposed that the ability of TNF to inhibit cAMP synthesis in microglia may exacerbate its response and contribute to cell damage in neuroinflammation and neurodegeneration, possibly through enhanced release of proinflammatory and/or cytotoxic factors.

Adenylyl Cyclases↗
PubMed2005

Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons.

There is good evidence that the kynurenine pathway (KP) and one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neurological diseases. While QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the capacity of astrocytes and neurons to produce QUIN is controversial. Using interferon gamma (IFN-gamma)-stimulated primary cultures of human mixed brain cells, we assayed expression of the KP regulatory enzyme indoleamine 2,3-dioxygenase (IDO) and QUIN production by immunocytochemistry. Using IFN-gamma-stimulated purified cultures of neurons, astrocytes, microglia and macrophages, we studied IDO expression by RT-PCR and production of QUIN using mass spectrometry. We found that astrocytes, neurons, and microglia expressed IDO but only microglia were able to produce detectable amounts of QUIN. However, astrocytes and neurons had the ability to catabolize QUIN. This study also provides the first evidence of IDO expression and lack of production of QUIN in culture of primary human neurons.

Astrocytes↗
PubMed2005

Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia.

Microglia are the major cell type involved in neuroinflammatory events in brain diseases such as encephalitis, stroke, and neurodegenerative disorders, and contribute significantly to the release of prostaglandins (PGs) during neuronal insults. In this report, we studied the immediate-early intracellular signalling pathways in microglia, following bacterial lipopolysaccharide (LPS) stimulation, leading to the synthesis and release of PGE2. Here we show that LPS induces cyclooxygenase (COX) 2 by activating sphingomyelinases leading to the release of ceramides, which in turn, activate the p38 mitogen-activated protein kinases (MAPK), but not the p42/44 MAPK. We further show that exogenously added ceramide analogue (C2-ceramide) also induce PGE2 synthesis through a p38 MAPK-dependent pathway. This potential nature of ceramides in activating microglia suggests that endogenously produced ceramides during neuronal apoptosis in ischemia or neurodegenerative diseases could also contribute to the amplification of neuroinflammatory events. In contrast to protein kinase C (PKC) and phosphocholine-specific phospholipase C (PC-PLC), which transcriptionally regulate LPS-induced COX-2 synthesis, inhibition of phospholipase A2 (PLA2) has no effect on COX-2 transcription, although it inhibits the release of PGE2. Transcriptional regulation of LPS-induced COX-2 by PKC is further proved by the ability of the PKC inhibitor, Gö 6976, to inhibit LPS-induced 8-isoprostane synthesis, but not affecting LPS-induced COX-2 activity. Our data with 8-isoprostane also indicates that COX-2 plays a major role in ROS production in LPS-activated microglia. This detailed view of the intracellular signaling pathway in microglial activation and COX-2 expression opens a new therapeutic window in the search for new and more effective central anti-inflammatory agents.

Animals↗
PubMed2006

Alpha-tocopherol (vitamin E) induces rapid, nonsustained proliferation in cultured rat microglia.

Microglial cells undergo cell division in vitro, as well as in vivo after brain injury. Mitotic activity of microglia suggests that they have limited life spans and rely on self-renewal to replace senescent cells. In the current study we examined long-term effects of antioxidants vitamin E and alpha-lipoic acid on cultured rat microglia with respect to proliferative ability, telomere length, telomerase activity, and interleukin-1beta (IL-1beta) production. We report that vitamin E induces dramatic microglial proliferation, as measured by MTT assay and BrdU incorporation, surpassing that of the well-known microglial mitogen granulocyte macrophage-colony stimulating factor, and therefore establishing vitamin E as the most potent, known mitogen for microglia in vitro. The high rate of microglial proliferation resulted in a concomitant decrease in telomere length and telomerase activity. Production of IL-1beta was significantly decreased in vitamin E-treated microglia in vitro. Our findings provide an impetus to investigate potential benefits of vitamin E supplementation on microglial renewal capacity in vivo during aging or after brain injury.

Animals↗
PubMed2006

Microglia instruct subventricular zone neurogenesis.

Microglia are increasingly implicated as a source of non-neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self-renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia-conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self-renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.

Animals↗
PubMed2007

Upregulation of CCAAT/enhancer binding protein beta in activated astrocytes and microglia.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPbeta in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPbeta expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPbeta in untreated mixed glial cultures. C/EBPbeta is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS-induced upregulation of glial C/EBPbeta is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPbeta nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS-induced increase in microglial C/EBPbeta is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPbeta as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPbeta expression in activated glial cells in vivo. These findings support a role for C/EBPbeta in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPbeta-regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation. (c) 2006 Wiley-Liss, Inc.

Animals↗
PubMed2007

Evidence for synaptic stripping by cortical microglia.

Recent studies have described significant demyelination and microglial activation in the cerebral cortex of brains from multiple sclerosis patients. To date, however, experimental models of cortical demyelination or cortical inflammation have not been extensively studied. In this report we describe focal cortical inflammation induced by stereotaxic injection of killed bacteria (BCG), followed 1 month later by subcutaneous injection of the same antigen, a protocol that overcomes the immune privilege of the cortex. Intracerebral BCG injection produced focal microglial activation at the injection site (termed acute lesion). Ten days after peripheral challenge (termed immune-mediated lesion), larger areas and higher densities of activated microglia were found near the injection site. In both paradigms, activated microglia and/or their processes closely apposed neuronal perikarya and apical dendrites. In the immune-mediated lesions, approximately 45% of the axosomatic synapses was displaced by activated microglia. Upon activation, therefore, cortical microglial migrate to and strip synapses from neuronal perikarya. Since neuronal pathology was not a feature of either the acute or immune-mediated lesion, synaptic stripping by activated microglia may have neuroprotective consequences.

Animals↗
PubMed2007

Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis.

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1beta, TNF-alpha, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.

Animals↗
PubMed2001

From theory to therapy: implications from an in vitro model of ramified microglia.

Microglia are the principal immune cells in the central nervous system (CNS), characterized by a highly specific morphology and unusual antigenic phenotype. An increasing number of studies have focused on the role of microglia in the pathogenesis of neurodegenerative diseases. To elucidate the function of microglial cells under several neuropathological conditions, we have studied and established a cell culture model that allows us to cultivate microglial cells in their inactive, resting (ramified) phenotype. In the first part of this work, we describe the interaction of microglia cells with their epithelial (astrocytic) microenvironment. The second part reviews experiments with microglia cell cultures to elucidate underlying signalling pathways and summarizes recent advances of our knowledge in microglial molecular pathways that may ultimately lead to neurodegeneration.

Animals↗
PubMed2001

Neurotrophin secretion from cultured microglia.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.

Animals↗
PubMed2005

Cyclopentenone prostaglandins PGA2 and 15-deoxy-delta12,14 PGJ2 suppress activation of murine microglia and astrocytes: implications for multiple sclerosis.

The cyclopentenone prostaglandin (cPG) 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) has been identified as a potent antiinflammatory agent that is able to inhibit the activation of macrophages and microglia. Additionally, 15d-PGJ(2) is able to ameliorate the clinical manifestations of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Many biological effects of 15d-PGJ(2) have been attributed to the peroxisome proliferator activated receptor-gamma (PPAR-gamma). PGA(2), like 15d-PGJ(2), is a cPG. The aim of this study is to compare the relative effectiveness of these two cPGs in inhibiting the inflammatory response of mouse microglia and astrocytes, two cell types that upon activation may contribute to the pathology of EAE and MS. Purified primary mouse microglia and astrocytes were treated with either 15d-PGJ(2) or PGA(2) and then stimulated with either lipopolysaccharide (LPS) or a combination of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. The results show that 15d-PGJ(2) and PGA(2) both potently inhibited the production of nitrite, as well as proinflammatory cytokines and chemokines, from microglia and astrocytes. Generally, regulation of NO production was more sensitive to 15d-PGJ(2), however, cytokine and chemokine production was more sensitive to PGA(2) treatment. These results demonstrate for the first time that PGA(2) is a potent antiinflammatory mediator.

Animals↗
PubMed2005

Microglia function in brain tumors.

Microglia play an important role in inflammatory diseases of the central nervous system (CNS). These cells have also been identified in brain neoplasms; however, as of yet their function largely remains unclear. More recent studies designed to characterize further tumor-associated microglia suggest that the immune effector function of these cells may be suppressed in CNS tumors. Furthermore, microglia and macrophages can secrete various cytokines and growth factors that may contribute to the successful immune evasion, growth, and invasion of brain neoplasms. A better understanding of microglia and macrophage function is essential for the development of immune-based treatment strategies against malignant brain tumors.

Animals↗
PubMed2001

Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia.

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).

Amyloid beta-Peptides↗
PubMed1999

Microglia exhibit clonal variability in eliciting cytotoxic T lymphocyte responses independent of class I expression.

Microglia are important immunoregulatory cells within the central nervous system (CNS). Viral infection of primary microglia and splenic macrophage clones revealed that both exhibited a heterogeneous, but relatively low, sensitivity to cytolysis mediated by CD8(+) cytotoxic T lymphocytes (CTL). The majority of clones were poor in processing and presenting epitopes, despite triggering lysis when coated with peptide. These characteristics were retained by stable microglia lines. Reduced lysis did not correlate with class I expression and IFN-gamma treatment only partially enhanced recognition. In contrast, targeting the epitope into the endoplasmic reticulum restored cytolysis to levels achieved with exogenous peptide. An inherent resistance to cytolysis was revealed by efficient engagement of T cells in competition assays and the inability of saturating peptide to enhance cytolysis. These data suggest that microglia heterogeneity in antigen processing, in addition to low sensitivity to CTL lysis, contributes to limited CD8(+) T cell responses and viral CNS persistence.

Animals↗
PubMed1996

Conditioned medium from activated microglia promotes cholinergic differentiation in the basal forebrain in vitro.

In earlier studies we found that treatment with interferon-gamma (IFN-gamma) produced an 8- to 11-fold increase in choline acetyltransferase (ChAT) in cultured cells taken from Embryonic Day 16 (E16) septal nuclei with adjacent basal forebrain (SN/BF). Since younger cultures responded even more profoundly to IFN treatment, we have tested the possibility that the action of IFN (or its intermediate; see below) is to prompt the cholinergic differentiation of neuronal precursors. SN/BF cultures of various ages were labeled with a retrovirus engineered to express beta-galactosidase (Lac-Z), and ChAT-positive descendants of the retrovirally labeled precursors were counted. IFN-gamma treatment of cultures caused as much as an 8.8-fold increase in the proportion of ChAT-positive cells present in Lac-Z-positive clones, suggesting that IFN promoted cholinergic differentiation in precursor populations. By contrast, bFGF increased clone size but did not change the proportion of ChAT-positive cells. NGF affected neither. Only ameboid microglia present in the cultures responded to IFN with characteristic nuclear translocation of the signal transducing molecule p91, suggesting that a microglial-derived molecule may mediate the action of IFN. Consistent with this hypothesis, conditioned media from cultures of enriched, activated microglia also increased ChAT activity in a dose-dependent fashion. Conditioned media from an unstimulated macrophage/monocyte cell line (RAW 264.7) also proved extremely efficacious in raising ChAT activity. In addition, conditioned media from both activated microglia and RAW 264.7 cells increased the proportion of ChAT-positive cells in retrovirally labeled clones to the same extent as IFN itself, suggesting the possibility that they contain the molecule(s) that mediates the action of IFN. Preliminary characterization of this molecule suggests that it is a very stable and large protein. Together these data suggest that a molecule promoting cholinergic differentiation is produced by activated microglia and other macrophage-like cells. The identity of this molecule and its precise role in normal development await its further purification.

Animals↗
PubMed1997

Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro.

This study examined the regulation of apolipoprotein E (apoE) by 17beta-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as an in vivo model and cultured astrocytes and mixed glia as in vitro models. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA. In vitro, E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens. In situ hybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microglia in vivo and the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimer's disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.

Animals↗
PubMed1997

Microglia responses in the CNS following sciatic nerve transection in C57BL/Wld(s) and BALB/c mice.

The present study employed C57BL/Wld(s) mice to investigate whether a delay in microglia reaction would occur similar to the delay that occurs in macrophage response after sciatic neurectomy. The results were compared with control BALB/c mice. The observations showed that in both strains of mice there was no delayed microglia response around lesioned motoneurons and around the central processes of the dorsal root ganglion cells after sciatic neurectomy in the adult. The increased Mac-1 staining appeared as early as 1 day postoperation (dpo). This indicates that microglial cells and macrophages respond to different signals generated by neurectomy. In both strains of mice, the number of microglia in the neonate was much less than that in the adult and the increase in Mac-1 staining was detectable only at 3 dpo in both strains of mice. A significant loss of motoneurons was detected after sciatic neurectomy in the neonate. However, there were no significant differences in the mean percentages of motoneuron loss between the two strains of mice at 5, 10, and 15 dpo. It is surmised that the lack of an adequate number of mature microglia in the neonates and their tardy expression of CR3 antigenicity may contribute to the motoneuron loss.

Aging↗
PubMed2000

A serum factor enhances production of nitric oxide and tumor necrosis factor-alpha from cultured microglia.

The pathological activation of microglia has been implicated in ischemic neuronal damage and some neurodegenerative diseases; however, the mechanism of microglial activation is not well understood. Previously, we showed that a serum factor, albumin, increased O(2)(-) production by cultured microglia (Si et al., 1997, Glia 21: 413-418). In the present study, we found that serum also enhanced lipopolysaccharide (LPS)-induced production of nitric oxide and tumor necrosis factor-alpha, which are other important neurotoxins released by activated microglia. In the presence of 0.1% normal rat serum, the half-effective concentration for LPS decreased from 300 to 1 ng/ml. The factor seemed to be a relatively high-molecular-weight protein because the factor was retained after a molecular sieve (50 kDa) membrane separation. The factor was labile to trypsinization and heat treatment at 72 degrees C for 5 min but was stable at 56 degrees C for 60 min. Several purified serum proteins including albumin could not mimic the enhancing effect of serum. Acute-phase serum showed a potent enhancing effect at a 10 times lower concentration than the normal serum. By gel filtration chromatography, the enhancing effect observed was a single peak at about 60 kDa. These results suggest that some serum protein infiltrates into brain parenchyma after blood-brain barrier disruption and such protein may result in neuronal damage by activating microglia to release neurotoxins in some central nervous system diseases.

Animals↗