Murine Microglial Cell Line BV-2 Research Articles

Dopamine inhibits lipopolysaccharide-induced nitric oxide production through the formation of dopamine quinone in murine microglia BV-2 cells

Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D1-like and D2-like DA receptor antagonists, respectively. In addition, pretreatment with (−)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208–243) and bromocriptine, D1-like and D2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells.

Investigation on Lipopolysaccharide Activated Microglia by Phosphoproteomics and Phosphoinositide Lipidomics

Microglia are the confined immune cells of the central nervous system (CNS). In response to injury or infection, microglia readily become activated and release proinflammatory mediators that are believed to contribute to microglia-mediated neurodegeneration. In the present study, inflammation was induced in the immortalized murine microglial cell line BV-2 by lipopolysaccharide (LPS) treatment. We firstly performed phosphoproteomics analysis and phosphoinositide lipidomics analysis with LPS activated microglia in order to compare phosphorylation patterns in active and inactive microglia and to detect the pattern of changes in phosphoinositide regulation upon activation of microglia. Mass spectrometry analysis of the phosphoproteome of the LPS treatment group compared to that of the untreated control group revealed a notable increase in the diversity of cellular phosphorylation upon LPS treatment. Additionally, a lipidomics analysis detected significant increases in the amounts of phosphoinositide species in the LPS treatment. This investigation could provide an insight for understanding molecular mechanisms underlying microglia-mediated neurodegenerative diseases.

Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

Constitutive and functional expression of runt-related transcription factor-2 by microglial cells

Runt-related transcription factor-2 (Runx2) is the master regulator of osteoblastogenesis with an ability to promote differentiation of mesenchymal stem cells into the osteoblastic lineage. We have previously shown constitutive and functional expression of Runx2 by astroglial cells. In this study, we investigated the possible expression of Runx2 by both murine microglia and microglial cell line BV-2 cells. Runx2 expression was seen in cultured microglia and BV-2 cells, while sustained exposure to 1mM ATP led to a significant but transient increase in mRNA and corresponding protein expression of Runx2 within 24h. The increase in Runx2 expression was invariably prevented by several chemicals with antagonistic properties for P2X7 purinergic receptor, calmodulin and calcineurin in BV-2 cells, with a P2X7 receptor agonist more than quadrupling Runx2 expression. A significant increase in Runx2 expression was seen in osteoclastic cells, but not in osteoblastic or chondrocytic cells, when exposed to a high concentration of ATP. In BV2-cells with control siRNA, a significant decrease was found in the number of cells with at least one process within 3h after the exposure to 1mM ATP, followed by an increase up to 24h. However, Runx2 siRNA significantly deteriorated the property to induce delayed process extension during 6–24h after exposure to ATP along with drastically decreased Runx2 protein levels. These results suggest that Runx2 is constitutively and functionally expressed by microglial cells with responsiveness to ATP for upregulation in the murine brain.

Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS

Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxic concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IкB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation.

Amyloid precursor-like protein 2 C-terminal fragments upregulate S100A9 gene and protein expression in BV2 cells.

The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secreting inflammatory cytokines, and is an important target in the treatment of nerve inflammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 (APLP2) C-terminal fragments (CTFs; C57, C50 and C31) in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein expression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection in Alzheimer's disease.

Inflammation induces multinucleation of Microglia via PKC inhibition of cytokinesis, generating highly phagocytic multinucleated giant cells

Microglia are brain macrophages, which can undergo multinucleation to give rise to multinucleated giant cells that accumulate with ageing and some brain pathologies. However, the origin, regulation and function of multinucleate microglia remain unclear. We found that inflammatory stimuli, including lipopolysaccharide, amyloid β, α-synuclein, tumour necrosis factor-α and interferon γ, but not interleukin-4, induced multinucleation of cultured microglia: primary rat cortical microglia and the murine microglial cell line BV-2. Inflammation-induced multinucleation was prevented by a protein kinase C (PKC) inhibitor Gö6976 (100 nM) and replicated by a PKC activator phorbol myristate acetate (160 nM). Multinucleation was reversible and not because of cell fusion or phagocytosis, but rather failure of cytokinesis. Time-lapse imaging revealed that some dividing cells failed to abscise, even after formation of long cytoplasmic bridges, followed by retraction of bridge and reversal of cleavage furrow to form multinucleate cells. Multinucleate microglia were larger and 2-4 fold more likely to phagocytose large beads and both dead and live PC12 cells. We conclude that multinucleate microglia are reversibly generated by inflammation via PKC inhibition of cytokinesis, and may have specialized functions/dysfunctions including the phagocytosis of other cells. Inflammation resulted in the accumulation of multiple nuclei per cell in cultured microglia. This multinucleation was reversible and due to a PKC-dependent block of the last step of cell division. Multinucleate microglia were larger and had a greater capacity to phagocytose other cells, suggesting they might remove neurons in the brain.

Global gene expression changes in BV2 microglial cell line during rabies virus infection

Microglia plays a crucial role during virus pathogenesis in the central nervous system (CNS). Infection by rabies virus (RABV) causes a fatal infection in the CNS of all warm-blooded animals. However, the microglial responses to RABV infection have been scarcely reported. To better understand microglia–RABV interactions at the transcriptional level, a genome wide gene expression profile in mouse microglial cells line BV2 was performed using microarray analysis. The global messenger RNA changes in murine microglial cell line BV2 after 12, 24 and 48h of infection with rabies virus CVS-11 strain were investigated using DNA Microarray and quantitative real-time PCR. Infection of CVS-11 at different time points induced different gene expression signatures in BV2 cells. The expression patterns of differentially expressed genes are shown by K-means clustering in four clusters in RABV- or mock-infected microglia at 12, 24 and 48h post infection (hpi). Gene ontology and network analysis of the differentially expressed genes in responses to RABV were performed by the Ingenuity Pathway Analysis system (IPA, Ingenuity® Systems, http://www.ingenuity.com). The results revealed that 28 genes were significantly up-regulated (P<0.01) and 1 gene was significantly down-regulated (P<0.01) in microglial cells at 12hpi, 72 genes were significantly up-regulated (P<0.01) and 24 genes were significantly down-regulated (P<0.01) at 24hpi, and 671 genes were significantly up-regulated (P<0.01) and 190 genes were significantly down-regulated (P<0.01) at 48hpi. Genes in BV2 were significantly regulated (P<0.01) in response to RABV infection and they were found to be interferon stimulated genes (Isg15, Isg20, Oasl1, Oasl2, Ifit2, Irf7 and Ifi203), chemokine genes (Ccl5, Cxcl10 and Ccrl2) and the proinflammatory factor gene (Interleukin 6). The results indicated that the differentially expressed genes from microglial cells after RABV infection were mainly involved in innate immune responses, inflammatory responses and host antiviral responses.

Rho Kinase Inhibitor Fasudil Regulates Microglia Polarization and Function

Macrophages/microglia exhibit phenotypic and functional heterogeneity under physiological and pathological conditions. Owing to this heterogeneity, the polarization of macrophages/microglia is capable of effecting both detrimental and beneficial outcomes in various disease processes. In this study, murine microglial cell line BV-2 and primary microglia were used as cell models to elucidate the polarization of microglia. Using flow cytometry, Western blot, chemical/enzymatic determination, and immunohistochemistry, treatment with LPS primed microglia into the M1 phenotype in both BV-2 cells and primary microglia, while fasudil skewed LPS-stimulated M1 toward M2 microglia, which showed lower NF-κB activity and inflammatory cytokines IL-1ß, IL-6, and TNF-a, and increased anti-inflammatory cytokine IL-10. To examine whether the regulatory role of LPS and fasudil on microglia can occur in vivo, mice were administered LPS (25 µg/10 µl) via nasal instillation every other day for 1 month. The results demonstrated that LPS also triggered iNOS⁺/CD11b⁺ M1 microglia in the brain, while fasudil increased Arg-1⁺/CD11b⁺ M2 microglia, although the difference did not reach statistical significance. Fasudil-conditioned microglia medium promoted a neuroprotective effect against PC12 neurons, suggesting that fasudil-induced M2 microglia contribute to the survival of neurons. These results indicate a new treatment option whereby fasudil inhibits the inflammatory response by controlling a helpful polarization in microglia/macrophages.

Anti-inflammatory homoisoflavonoids from the tuberous roots of Ophiopogon japonicus

Two new homoisoflavonoids, named ophiopogonone E (1) and ophiopogonanone H (2), together with thirteen known ones (3–15) were isolated from the tuberous roots of Ophiopogon japonicus. Their structures were elucidated by spectroscopic and chemical analyses. Compounds 7 and 15 were isolated from the genus for the first time. In addition, compounds 2–15 were evaluated for their effects on the inhibition of NO production induced by lipopolysaccharide in the murine microglial cell line BV-2. Compounds 2, 4, 6, 7, 10, 11 showed potent inhibitory effects on NO production with IC50 values of 20.1, 17.0, 7.8, 5.1, 19.2 and 14.4μM respectively.

Selective Upregulation of Per1 mRNA Expression by ATP Through Activation of P2X7 Purinergic Receptors Expressed in Microglial Cells

Clock genes are believed to play a pivotal role in the generation and oscillation of circadian rhythm as a central clock in the hypothalamic suprachiasmatic nucleus in the mammalian brain. In this study, mRNA expression was for the first time demonstrated with clock genes in both cultured murine microglia and microglial cell line BV-2 cells. Exposure to ATP transiently increased Period-1 (Per1) mRNA expression without affecting that of other clock genes in BV-2 cells, while a similarly transient increase was shown in Per1 mRNA expression in a manner sensitive to P2X7 purinergic receptor antagonists in cultured microglia exposed to ATP. In BV-2 cells transfected with a Per1 promoter luciferase reporter plasmid, ATP significantly increased luciferase activity in a manner sensitive to a P2X7-receptor antagonist. In both microglia and BV-2 cells, a significant increase by ATP was seen in the immunocytochemical fluorescence intensity of cells expressing Per1 protein, with mRNA expression of different P2 receptors including P2X7. Per1 siRNA significantly decreased the number of cells with processes in BV-2 cells exposed to ATP. These results suggest that ATP selectively promotes Per1 expression through gene transactivation after stimulation of P2X7 purinergic receptors in microglial cells.

Abscisic acid does not evoke calcium influx in murine primary microglia and immortalised murine microglial BV-2 and N9 cells

Brain microglia are resident macrophage-like cells representing the first and main form of active immune response during brain injury. Microglia-mediated inflammatory events in the brain are known to be associated with chronic degenerative diseases such as Multiple Sclerosis, Parkinson’s, or Alzheimer’s disease. Therefore, identification of mechanisms activating microglia is not only important in the understanding of microglia-mediated brain pathologies, but may also lead to the development of new anti-inflammatory drugs for the treatment of chronic neurodegenerative diseases. Recently, abscisic acid (ABA), a phytohormone regulating important physiological functions in higher plants, has been proposed to activate murine microglial cell line N9 through increased intracellular calcium. In the present study, we determined the response to ABA and its analogues from murine primary microglia and immortalized murine microglial cell line BV-2 and N9 cells. A Fura-2-acetoxymethyl ester (Fura-2AM)-based ratiometric calcium imaging and measurement technique was used to determine the intracellular calcium changes in these cells when treated with (−)-ABA, (+)-ABA, (−)- trans-ABA and (+)- trans-ABA. Both primary microglia and microglial cell lines (BV-2 and N9 cells) showed significant increase in intracellular calcium ([Ca 2+]i) in response to treatment with ATP and ionomycine. However, ABAs failed to evoke dose- and time-dependent [Ca 2+]i changes in mouse primary microglia, BV-2 and N9 cells. Together, these surprising findings demonstrate that, contrary to that reported in N9 cells [3], ABAs do not evoke intracellular calcium changes in primary microglia and microglial cell lines. The broad conclusion that ABA evokes [Ca 2+]i in microglia requires more evidence and further careful examination.

The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation

The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies.

CAMP mediates ammonia‐induced programmed cell death in the microglial cell line BV‐2

Although ammonia is a well-known neuropathogenic factor, the cellular mechanisms of ammonia toxicity are less characterized. Up to now, the main focus of ammonia toxicity has been on astrocytes and neurons. Despite the significance of microglia in neurodegenerative diseases, little is known about their responsiveness to ammonia. In the present study, we found that ammonia triggered mitosis at concentrations between 30 microm and 3.0 mm but apoptosis at concentrations >or= 1.0 mm in the murine microglial cell line BV-2. Most apoptotic cells showed an accumulation of condensed chromatin at the nuclear envelope, blebbing of the plasma membrane, formation of apoptotic bodies and an increase in caspase 3/7 activity. Blockade of caspase 3/7 activity by Ac-DEVD-CHO suppressed ammonia-induced apoptosis. Surprisingly, some BV-2 cells exposed to ammonia displayed clear signs of mitotic catastrophe, a type of cell death occurring during mitosis. In a further series of experiments, we found that cyclic adenosine 3',5'-monophosphate (cAMP) mediated the apoptogenic effects of ammonia, because (i) ammonia dose-dependently elevated the intracellular cAMP level, (ii) blockade of the adenylyl cyclase by SQ-22536 suppressed ammonia-induced apoptosis, (iii) inhibition of phosphodiesterases (PDEs) by the nonselective PDE inhibitor IBMX, or by the PDE4-selective inhibitor rolipram, increased the relative number of apoptotic cells, and (iv) the cAMP analogues 8-bromoadenosine cAMP and Sp-cAMP mimicked the effect of ammonia and triggered apoptosis. Taken together, our results indicate that distinct concentrations of ammonia trigger opposite signalling pathways in microglial cells.

DIFFERENCES BETWEEN LIPOPOLYSACCHARIDE AND DOUBLE-STRANDED RNA IN INNATE IMMUNE RESPONSES OF BV2 MICROGLIAL CELLS

Microglial cells are thought to be major inflammatory cells in the central nervous system; however, sufficient information about the effects of double-stranded RNA (dsRNA) in microglial cells is lacking. The present study compared the innate immune responses of the murine microglial cell line BV2 to dsRNA and lipopolysaccharide (LPS). It showed that the effect of dsRNA was similar to that of LPS treatment. The dsRNA induced several pro-inflammatory factors such as TNF-α, IL-6, IL-1β, and IL-1Ra. Furthermore, the expression level of COX-2 was increased after treatment with dsRNA. However, the induction level of IL-1β by dsRNA was less than those of the other cytokines that were measured. These results suggest that, although both dsRNA and LPS trigger pro-inflammatory responses, the intracellular signaling pathway and inflammation pattern of dsRNA and LPS may be different. Therefore, dsRNA produced during viral infection could precipitate neurological abnormalities through chronic inflammation.

Oxidative Toxicity in BV‐2 Microglia Cells: Sesamolin Neuroprotection of H2O2 Injury Involving Activation of p38 Mitogen‐Activated Protein Kinase

Reactive oxygen species (ROS) has been proposed to play a pathogenic role in neuronal injury. Sesame antioxidants that inhibit lipid peroxidation and regulate cytokine production may suppress ROS generation. In this study, we focused on the effect of sesamolin on H2O2-induced neurotoxicity and ROS production in the murine microglial cell line BV-2. Results indicate that the H2O2 elicited BV-2 cell death in a concentration- and time-dependent manner. ROS generation in BV-2 cells was time-dependently increased by the H2O2 treatment. Sesamolin reduced ROS generation in BV-2 cells. p38 mitogen-activated protein kinase (MAPK) and caspase-3 were also activated in BV-2 cells under H2O2 stress. Sesamolin was able to inhibit H2O2-induced p38 MAPK and caspase-3 activation and cell death. In addition, sesamolin preserved superoxide dismutase and catalase activities in BV-2 cells under H2O2 stress. In conclusion, sesamolin protects microglia against H2O2-induced cell injury and this protective effect was accompanied by its inhibition of p38 MAPK and caspase-3 activation and ROS production.

S-Nitrosoglutathione Reduces Inflammation and Protects Brain against Focal Cerebral Ischemia in a Rat Model of Experimental Stroke

Preservation of endothelial functions with low-dose nitric oxide (NO) and inhibition of excessive production of NO from inducible NO synthase (iNOS) is a potential therapeutic approach for acute stroke. Based on this hypothesis, an NO modulator, S-nitrosoglutathione (GSNO) was used, which provided neuroprotection in a rat model of focal cerebral ischemia. Administration of GSNO after the onset of ischemia reduced infarction and improved cerebral blood flow. To understand the mechanism of protection, the involvement of inflammation in ischemic brain injury was examined. Treatment with GSNO reduced the expression of tumor necrosis factor-alpha, interleukin-1beta, and iNOS; inhibited the activation of microglia/macrophage (ED1, CD11-b); and downregulated the expression of leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 in the ischemic brain. The number of apoptotic cells (including neurons) and the activity of caspase-3 were also decreased after GSNO treatment. Further, the antiinflammatory effect of GSNO on expression of iNOS and activation of NF-kappaB machinery in rat primary astrocytes and in the murine microglial cell line BV2 was tested. Cytokine-mediated expression of iNOS and activation of NF-kappaB were inhibited by GSNO treatment. That GSNO protects the brain against ischemia/reperfusion injury by modulating NO systems, resulting in a reduction in inflammation and neuronal cell death was documented by the results.

Ibuprofen and apigenin induce apoptosis and cell cycle arrest in activated microglia

In case of injury or disease, microglia are recruited to the site of the pathology and become activated as evidenced by morphological changes and expression of pro-inflammatory cytokines. Evidence suggests that microglia proliferate by cell division to create gliosis at the site of pathological conditions such as the amyloid plaques in Alzheimer's disease and the substantia nigra of Parkinson's disease patients. The hyperactivation of microglia contributes to neurotoxicity. In the present study we tested the hypothesis that anti-inflammatory compounds modulate the progression of cell cycle and induce apoptosis of the activated cells. We investigated the effects of ibuprofen (non-steroidal anti-inflammatory drug) and apigenin (a flavonoid with anti-inflammatory and anti-proliferative properties) on the cell cycle of the murine microglial cell line BV-2. The findings indicate that apigenin-induced cell cycle arrest preferentially in the G2/M phase and ibuprofen caused S phase arrest. The binding of annexin V–FITC to the membranes of cells which indicates the apoptotic process were examined, whereas the DNA was stained with propidium iodide. Both apigenin and ibuprofen induced apoptosis significantly in early and late stages. The induction of apoptosis by ibuprofen and apigenin was confirmed using TUNEL assay, revealing that 25 μM apigenin and 250 μM ibuprofen significantly increased apoptosis in BV-2 cells. The results from the present study suggest that anti-inflammatory compounds might inhibit microglial proliferation by modulating the cell cycle progression and apoptosis.

Estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor beta.

Estrogens are well known to exert antiinflammatory effects outside the central nervous system (CNS). They have also been shown to exert neuroprotective effects in the CNS after several types of injury, including neurodegeneration. However, the molecular mechanisms by which these effects occur remain unclear. Because microglial hyperactivation and their production of neurotoxins is associated with many types of brain injury for which estrogens are beneficial, we sought to investigate the ability of estrogen to modulate microglial function. Furthermore, because little is known regarding the role of each of the two known estrogen receptors (ERs) in microglia, our studies were designed to test the hypothesis that 17beta-estradiol (E(2)) exerts antiinflammatory effects in microglia, specifically via interactions with ERbeta. We tested this hypothesis using the murine microglial cell line BV-2, which naturally expresses only ERbeta. Our results indicate that not only does E(2) decrease lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, it also reduces the expression of cyclooxygenase-2, a target for estrogen that has not previously been reported for ERbeta. We also observed that LPS-stimulated TNFalpha mRNA was increased by estrogen. E(2) exerts these effects within 30 min compared with typical estrogen transcriptional responses. Tamoxifen and ICI 182,780 differentially blocked the inhibitory effects of E(2) on LPS-stimulated iNOS and cyclooxygenase-2. In addition, we show that E(2) alters LPS-stimulated MAPK pathway activation, supporting the idea that alterations in the MAPKs may be a potential mechanism by which ERbeta mediates decreased microglial activation.

Galanin down-regulates microglial tumor necrosis factor-α production by a post-transcriptional mechanism

The neuropeptide galanin (GAL) is up-regulated following neuronal axotomy or inflammation. Since other neuropeptides act as immunomodulatory agents, we sought to determine whether GAL might affect the murine microglial cell line BV2, which expresses the GAL2 receptor. Even at very low concentrations, GAL inhibited tumor necrosis factor-alpha (TNFα) release but not TNFα mRNA levels in LPS-stimulated BV2 cells. Northern blot analysis showed that GAL inhibited the addition of a poly(A) tail, and stability assays showed that it also destabilized TNFα mRNA. Thus, GAL inhibits TNFα production by a post-transcriptional mechanism that both prevents the efficient addition of the poly(A) tail and accelerates TNFα mRNA degradation.