Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.     

Expression of Inducible Nitric Oxide Synthase and Fas/Fas Ligand Correlates with the Incidence of Apoptotic Cell Death in Atheromatous Plaques of Human Coronary Arteries

It was recently reported that inducible nitric oxide synthase was expressed in advanced atheromatous plaques. So we investigated the effect of NO or peroxynitrite reactive product of NO or O(2)(-) released by iNOS induced in macrophages or T lymphocytes on inflammatory cells in atheromatous plaques of human coronary arteries by immunohistochemistry. We found that iNOS was expressed in T lymphocytes and macrophages in T lymphocytes and macrophages coexisted advanced atheromatous areas. Most of the smooth muscle cells are not coexisted with T lymphocytes. We could not find iNOS in those smooth muscle cells. Only a small number of iNOS-positive smooth muscle cells were found close to T lymphocytes and macrophages. Markers for apoptotic cells induced in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed that many apoptotic T lymphocytes and macrophages existed near iNOS induced cells. Fas and Fas ligand were expressed in almost same areas that iNOS was expressed. By double-label immunostaining, Fas was expressed in T lymphocytes but Fas ligand was expressed in macrophages and in some T lymphocytes. These results suggest that NO from iNOS induces Fas and Fas ligand-mediated apoptosis and associates with regression of atherosclerosis. On the other hand, nitrotyrosine was detected wider areas than iNOS. So peroxynitrite from iNOS damages cells and tissues widely and may associate with progression of atherosclerosis. These results suggest an important role of iNOS in mediating both regressive changes and progressive change in atheromatous plaques.     

Modulation of NO and cytokines in microglial cells by Cu/Zn-superoxide dismutase.

The activation of microglial cells in response to neuropathological stimuli is one of the prominent features of human neurodegenerative diseases. Cytokines such as IL-1 beta and TNF-alpha and inflammation-related enzymes such as inducible nitric oxide synthase are usually induced during the activation of microglial cells. We investigated the modulation of the activation of microglial cell by transfecting a Cu/Zn-SOD cDNA into BV-2 cells. Parental and transfected BV-2 cells were then subjected to LPS stimulation. The results showed that in Cu/Zn-SOD-transfected BV-2 cells, the expression and activity of Cu/Zn-SOD increased. On the other hand, upon activation by LPS, these cells produced less NO, IL-1 beta, and TNF-alpha than the parental microglial cells. This finding suggests that superoxide may be an early signal triggering the induction of cytokines and that the transfected Cu/Zn-SOD may provide a neuroprotective function via suppression of microglial activation. In addition, this approach may provide a rationale for the development of treatments for neurodegenerative diseases.     

Differential regulation and function of Fas expression on glial cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.     

TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.     

Synthesis of 5α-cholestan-6-one derivatives and their inhibitory activities of NO production in activated microglia: Discovery of a novel neuroinflammation inhibitor

Glial activation-mediated neuroinflammation plays a pivotal role in the process of several neuroinflammatory diseases including stroke, Alzheimer’s diseases, Parkinson’s diseases, multiple sclerosis and ischemia. Inhibition of microglial activation may ameliorate neuronal degeneration under the inflammatory conditions. In the present study, a number of 5α-cholestan-6-one derivatives were prepared and the anti-inflammatory effects of these compounds were evaluated in LPS-stimulated BV-2 microglia cells. Those derivatives were synthesized from readily available hyodeoxycholic acid (1). Among the tested compounds, several analogs (16–18, 25, 35, 38) exhibited potent inhibitory activities on nitric oxide production with no or weak cell toxicity. Compound 16 also significantly suppressed the expression of TNF-α, interleukin (IL)-1β, cyclooxygenase (COX-2) as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 microglia cells. In addition, compound 16 markedly reduced infarction volume in a focal ischemic mice model.     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.     

Immunological characterization of C3H mice congenic for Fas(lprcg), C3h/HeJ-Fas(lprcg)/Fas(lprcg)

Fas(lpr) (lpr) and Fas(lprcg) (lpr(cg)) are allelic mutations of the Fas gene that is involved in apoptosis or programmed cell death. Lpr greatly reduces the expression of functional Fas and lpr(cg) expresses the death domain-disabled, non-functional Fas on the cell surface. C3H/HeJ mice congenic for lpr(cg) (C3H-lpr(cg)) were established and compared with C3H/HeJ-lpr/lpr (C3H-lpr) mice for their immunological and pathological features. Lymphadenopathy, splenomegaly, development of CD4- CD8- B220+ or double-negative (DN) T cells, renal pathology, and lymphoid cell infiltration in the lung and liver were not significantly different between C3H-lpr(cg) and C3H-lpr mice. Noticeably, however, the production of serum immunoglobulin, autoantibodies against double-strand DNA and serum immune complexes were significantly lower in C3H-lpr(cg) than in C3H-lpr mice. The results indicate that the death signal through the death domain of Fas is responsible for lymphoproliferation due to the accumulation of DN T cells and suggest that the region of Fas outside the death domain may be involved in autoantibody production. The newly-developed congenic C3H-lpr(cg) mice will provide a powerful tool for research into the function of Fas apart from apoptosis.     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA.     

Dehydroepiandrosterone inhibits lipopolysaccharide-induced nitric oxide production in BV-2 microglia

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimer's disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age-related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)-stimulated BV-2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS-stimulated BV-2 cell cultures. Pretreatment of BV-2 cells with DHEA reduced the LPS-induced iNOS mRNA and protein levels in a dose-dependent manner. The LPS-induced iNOS activity in BV-2 cells was decreased by the exposure of 100 microM DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS-stimulated BV-2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.     

Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.

Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumour necrosis factor alpha (TNFalpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNFalpha neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.     

A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents inflammation mediated neurotoxicity; role in microglial activation in BV-2 cells

A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory factors such as nitric oxide, prostaglandin E(2), tumor necrosis factor-α and interleukin-1β. Moreover, it suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2 cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be useful for treating neurodegenerative diseases related with neuroinflammation.     

Reactive nitrogen species-induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation. JNK activation by RNS was abolished in cells lacking functional Fas or following expression of a truncated version of Fas lacking the intracellular death domain. In contrast, RNS induced JNK potently in cells expressing a truncated version of tumor necrosis factor receptor 1 or cells lacking tumor necrosis factor receptor 1 (TNF-R1), illustrating a dependence of Fas but not TNF-R1 in RNS-induced signaling to JNK. Furthermore, Fas was oxidized, redistributed, and colocalized with Fas-associated death domain (FADD) in RNS-exposed cells, illustrating that RNS directly targeted Fas. JNK activation and cell death by RNS occurred in a Fas ligand- and caspase-independent manner. While the activation of JNK by RNS or FasL required FADD, the cysteine-rich domain 1 containing preligand assembly domain required for FasL signaling was not involved in JNK activation by RNS. These findings illustrate that RNS cause cell death in a Fas- and JNK-dependent manner and that this occurs through a pathway distinct from FasL. Thus, avenues aimed at preventing the interaction of RNS with Fas may attenuate tissue damage characteristic of chronic inflammatory diseases that are accompanied by high levels of RNS.     

Necrotic death pathway in Fas receptor signaling

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.     

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.     

Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture

Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.     

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip_L expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.     

Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death.