Kyoungho Suk

IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death.

Fas ligand (FasL), perforin, TNF-α, IL-1, and NO have been considered as effector molecule(s) leading to β cell death in autoimmune diabetes. However, the real culprit(s) in β cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-γ/TNF-α synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-γ and TNF-α, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-γ treatment conferred susceptibility to TNF-α-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-γ/TNF-α-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-γ/TNF-α-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-α-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-α Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-γ/TNF-α synergism is responsible for autoimmune diabetes in vivo as well as β cell apoptosis in vitro and suggest a novel signal transduction in IFN-γ/TNF-α synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.

NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells.

Abnormal activation of microglial cells has been implicated in various neurodegenerative diseases. Microglial activation needs to be tightly regulated for physiological maintenance and normal functioning of the central nervous system. Potential mechanisms for the down-regulation of activated microglial cells are the deactivation or elimination of activated cells. We hypothesized that the elimination of activated microglial cells by apoptosis is one of the key mechanisms of auto-regulation of activated microglial cells. To test this hypothesis, we utilized BV-2 mouse microglial cells and rat primary microglial cultures exposed to activating agents such as lipopolysaccharide and interferon-gamma, and investigated a possible correlation between apoptosis and activation of these cells. We found that the activation of microglial cells led to apoptotic death, and the activation state of microglial cells inversely correlated with cell viability. We have also demonstrated that: (i) NO was produced by activated microglial cells in a manner dependent on time and dose of activating agents; (ii) inhibition of NO synthesis by iNOS inhibitor blocked the apoptosis of activated microglial cells; (iii) an exogenous NO donor induced apoptosis of microglial cells; and (iv) inhibition of TNFalpha or FasL using neutralizing antibodies did not affect activation-induced apoptosis of microglial cells. These results indicated that activation of microglial cells leads to the production of NO, which in turn acts as the major mediator of cellular apoptosis in an autocrine fashion. Our work suggests the presence of auto-regulatory mechanism for microglial activation, which may have relevance in the pathogenesis of various neurodegenerative diseases possibly resulting from over-activation of microglial cells.

Hypoxia induces nitric oxide production in mouse microglia via p38 mitogen-activated protein kinase pathway

In vitro exposure of microglial cells to hypoxia induces cellular activation. Also, in vivo studies of glial activation following ischemic hypoxia have shown that neuronal cell death is followed by microglial activation. Thus, it is likely that toxic inflammatory mediators produced by activated microglial cells under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. Nitric oxide (NO), which is known to be produced by activated microglia, may participate in this process. In the current work, we sought to determine whether and how the production of NO and the expression of inducible NO synthase (iNOS) are triggered by hypoxia in microglial cells. Exposure of established microglial cell lines as well as primary mouse microglial cultures to mild hypoxia (8 h) followed by reoxygenation (24 h) induced the production of NO and TNFalpha, indicating that hypoxia could lead to the inflammatory activation of microglia. Hypoxic induction of NO was accompanied by iNOS induction. Moreover, hypoxia induced the activation of p38 MAPK, but not ERK or JNK/SAPK, in BV-2 mouse microglial cells. SB203580, a specific inhibitor of p38 MAPK, blocked the hypoxic induction of NO and iNOS. Taken together, our results indicated that hypoxia could induce inflammatory activation of microglia, and the hypoxic induction of NO production in microglia is mediated through p38 MAPK pathway. Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but may also promote neuronal injury indirectly via microglial activation.

The plant flavonoid wogonin suppresses death of activated C6 rat glial cells by inhibiting nitric oxide production

Flavonoids are a group of low molecular weight polyphenolic compounds derived from plants. 5,7-dihydroxy-8-methoxyflavone (Wogonin), a flavonoid originated from the root of Scutellaria baicalensis Georgi, has been shown to exert various anti-inflammatory effects such as inhibition of nitric oxide (NO) and prostaglandin E2 production in macrophages. Because glial cells have been previously shown to undergo NO-dependent apoptosis upon inflammatory activation and this auto-regulatory process may be negatively affected by exogenous factors possessing anti-inflammatory activities, we examined the effects of wogonin on NO production and activation-induced cell death of C6 rat glial cells. Activation of C6 glial cells with lipopolysaccharide (LPS), interferon-gamma, and tumor necrosis factor-alpha induced NO production followed by cell death. Pretreatment of C6 cells with wogonin before LPS and cytokine treatment dose-dependently inhibited NO production as well as death of activated C6 cells. Wogonin-mediated inhibition of NO production was accompanied by suppression of inducible nitric oxide synthase (iNOS) protein induction and nuclear factor kappa B (NF-kappaB) reporter activity. Wogonin, however, did not affect a NO donor-induced cytotoxicity. Taken together, our results indicate that wogonin inhibits activation-induced death of C6 glial cells by suppressing NO production, and these inhibitory effects of wogonin on NO production are exerted through inhibition of NF-kappaB-mediated iNOS induction.

Borrelia burgdorferi Gene Expression In Vivo and Spirochete Pathogenicity

ABSTRACT Borrelia burgdorferi spirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate of B. burgdorferiN40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. A B. burgdorferi genomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, including dbpAB,bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required for B. burgdorferi to effectively colonize the host, evade humoral responses, and cause disease.

Activation-induced cell death of rat astrocytes

Inflammatory activation of astrocytes has been implicated in various neurodegenerative diseases. The elimination of activated astrocytes by apoptosis or the deactivation may be the mechanisms for auto-regulation of activated astrocytes. To test the possibility of apoptotic elimination of activated astrocytes, we examined a potential correlation between activation state of astrocytes and their viability using C6 rat glial cells and rat primary astrocyte cultures exposed to a variety of inflammatory stimuli such as lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha. Nitric oxide production was measured to evaluate inflammatory activation of astrocytes. We found that: (i) the activation of astrocytes by the combination of lipopolysaccharide and inflammatory cytokines, but not by either alone, led to nitric oxide production followed by apoptotic cell death; (ii) the amount of nitric oxide produced by activated astrocytes was inversely proportional to the viability of the cells; (iii) inhibition of nitric oxide synthase by N-monomethyl L-arginine blocked death of activated astrocytes; and (iv) nitric oxide donors induced apoptosis of astrocytes in a caspase-dependent manner. Taken collectively, our results suggest that activated astrocytes produce nitric oxide as an autocrine mediator of caspase-dependent apoptosis, and this type of programmed cell death of astrocytes may be the underlying mechanism for the auto-regulation of inflammatory activation of astrocytes.

Neuroprotection by methanol extract of Uncaria rhynchophylla against global cerebral ischemia in rats

In traditional Oriental medicine, Uncaria rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Thus, in the current study, we evaluated neuroprotective effects of U. rhynchophylla after transient global ischemia using 4-vessel occlusion model in rats. Methanol extract of U. rhynchophylla administered intraperitoneally (100-1000 mg/kg at 0 and 90 min after reperfusion) significantly protected hippocampal CA1 neurons against 10 min transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxygenase-2 in hippocampus at 24 hr after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF-alpha and nitric oxide production in BV-2 mouse microglial cells in vitro. These anti-inflammatory actions of U. rhynchophylla extract may contribute to its neuroprotective effects.

Role of Antiproliferative B Cell Translocation Gene-1 as an Apoptotic Sensitizer in Activation-Induced Cell Death of Brain Microglia

Mouse brain microglial cells undergo apoptosis on exposure to inflammatory stimuli, which is considered as an autoregulatory mechanism to control their own activation. Here, we present evidence that an antiproliferative B cell translocation gene 1 (BTG1) constitutes a novel apoptotic pathway of LPS/IFN-γ-activated microglia. The expression of BTG1 was synergistically enhanced by LPS and IFN-γ in BV-2 mouse microglial cells as well as in primary microglia cultures. Levels of BTG1 expression inversely correlated with a proliferative capacity of the microglial cells. Tetracycline-based conditional expression of BTG1 not only suppressed microglial proliferation but also increased the sensitivity of microglial cells to NO-induced apoptosis, suggesting a novel mechanism of cooperation between LPS and IFN-γ in the induction of microglial apoptosis. An increase in BTG1 expression, however, did not affect microglial production of NO, TNF-α, or IL-1β, indicating that the antiproliferative BTG1 is important in the activation-induced apoptosis of microglia, but not in the activation itself. The synergistic action of LPS and IFN-γ in the microglial BTG1 induction and apoptosis was dependent on the Janus kinase/STAT1 pathway, but not IFN-regulatory factor-1, as demonstrated by a pharmacological inhibitor of Janus kinase (AG490), STAT1 dominant negative mutant, and IFN-regulatory factor-1-deficient mice. Taken together, antiproliferative BTG1 may participate in the activation-induced cell death of microglia by lowering the threshold for apoptosis; BTG1 increases the sensitivity of microglia to apoptogenic action of autocrine cytotoxic mediator, NO. Our results point out an important link between the proliferative state of microglia and their sensitivity to apoptogenic agents.

Hypoxic induction of caspase-11/caspase-1/interleukin-1β in brain microglia

Caspase-11 is an inducible protease that plays an important role in both inflammation and apoptosis. Inflammatory stimuli induce and activate caspase-11, which is required for the activation of caspase-1 or interleukin-1beta (IL-1beta) converting enzyme (ICE). Caspase-1 in turn mediates the maturation of proinflammatory cytokines such as IL-1beta, which is one of the crucial mediators of neurodegeneration in the central nervous system. Here, we report that hypoxic exposure of cultured brain microglia (BV-2 mouse microglia cells and rat primary microglial cultures) induces expression and activation of caspase-11, which is accompanied by activation of caspase-1 and secretion of mature IL-1beta and IL-18. Hypoxic induction of caspase-11 was observed in both mRNA and protein levels, and was mediated through p38 mitogen-activated protein kinase pathway. Transient global ischemia in rats also induced caspase-11 expression and IL-1beta production in hippocampus supporting our in vitro findings. Caspase-11-expressing cells in hippocampus were morphologically identified as microglia. Taken together, our results indicate that hypoxia induces a sequential event-caspase-11 induction, caspase-1 activation, and IL-1beta release-in brain microglia, and point out the importance of initial caspase-11 induction in hypoxia-induced inflammatory activation of microglia.

IFNα sensitizes ME‐180 human cervical cancer cells to TNFα‐induced apoptosis by inhibiting cytoprotective NF‐κB activation

Tumor necrosis factor α (TNFα) induces apoptosis of a variety of tumor cell types. The anti‐tumor effect of TNFα is often augmented by interferon (IFN) γ. We hypothesized that IFNα, which shares many biological activities with IFNγ, might also synergize with TNFα for the induction of tumor cell death. We tested our hypothesis using ME‐180 human cervical cancer cells exposed to either IFNα or TNFα alone or both. We analyzed the death of ME‐180 cells by biochemical and cytological means, and investigated the molecular mechanism underlying cytotoxic synergism between the two cytokines. We found that (i) IFNα/TNFα synergistically induced apoptosis of ME‐180 cells, which was accompanied by activation of caspases‐3 and ‐8; (ii) IFNα induced signal transducer and activator of transcription (STAT) 1 phosphorylation, and transfection of phosphorylation‐defective STAT1 dominant‐negative mutant inhibited IFNα/TNFα‐induced apoptosis; (iii) inhibition of nuclear factor κB (NF‐κB) by proteasome inhibitor MG‐132 sensitized ME‐180 cells to TNFα alone; (iv) IFNα treatment attenuated TNFα‐induced NF‐κB reporter activity, while it did not inhibit DNA binding of NF‐κB. Taken collectively, our results indicate that IFNα sensitizes ME‐180 cells to TNFα‐induced apoptosis by inhibiting TNFα‐mediated cytoprotective NF‐κB activation, and this sensitizing effect of IFNα is mediated through a STAT1‐dependent pathway.