Interleukin 1-β- and tumor necrosis factor-α-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity

Several adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM-1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), with IL-1β and TNF-α being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM-1 gene expression induced by the cytokines IL-1β and TNF-α. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 increase ICAM-1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM-1 gene expression inducible by IL-1β and TNF-α. Prolonged treatment of astrocytes with PMA results in a time-dependent downregulating of the PKC isoforms α, δ, and ?, and a concomitant diminution of ICAM-1 mRNA expression induced by IL-1β, TNF-α, and PMA itself at specific time points post-PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL-1β and TNF-α enhancement of ICAM-1 gene expression in rat astrocytes. © 1995 Wiley-Liss, Inc.     

Soybean isoflavone alleviates β-amyloid 1-42 induced inflammatory response to improve learning and memory ability by down regulation of Toll-like receptor 4 expression and nuclear factor-κB activity in rats

β-amyloid 1-42 (Aβ1-42)-induced learning and memory impairment in rats is believed to be associated with inflammation. Cytokine production is a key pathologic event in the progression of inflammatory processes.In this rat study, soybean isoflavones (SIF) was used to investigate it's protective effects on inflammation caused by β-amyloid 1-42 (Aβ1-42), which is associated with learning and memory impairment in Alzheimer disease. We characterized the learning and memory ability. cytokine profiles of circulating interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) in the serum and the expression of Toll like receptor4 (TLR4) and nuclear factor-κB p65 (NF-κB p65) mRNA and protein in the brain tissue following intracerebroventricular administration of Aβ1-42 by miniosmotic pump for 14 days. The results showed that functional deficits of learning and memory in SIF treatment groups were significantly improved compared to the control group without SIF treatment in water maze test. The serum IL-1β and TNF-α level were significantly increased, and the expressions of TLR4 and NF-κB p65 mRNA and protein in the brain were up-regulated, indicating inflammation response was initiated following administration of Aβ1-42. After intragastric pre-treatment with SIF, inflammatory cytokines was significantly reduced and also SIF reversed the Aβ1-42 induced up-regulation of TLR4 and NF-κB p65 mRNA and protein expression in the brain and expression of NF-κB p65 in nuclei. These results suggested that SIF reduced the cytokine cascade and inflammatory response induced by Aβ1-42 which could result in the improvement of spatial learning and memory ability impairment in the rats.     

Differential regulation by cytokines of human astrocyte nitric oxide production

Reactive nitrogen intermediates, such as nitric oxide (NO), play an important role in host-defense and injury. Human astrocytes released abundant NO upon stimulation with the pro-inflammatory cytokine interleukin (IL)-1β, which was potentiated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. IL-1 receptor antagonist protein markedly attenuated astrocyte NO production. The anti-inflammatory cytokines IL-4 and IL-10 potently suppressed IL-1β plus IFN-γ-stimulated NO, while transforming growth factor-β preferentially inhibited IL-1β plus TNF-α-stimulated production of NO. These findings suggest that while IL-1 plays a key role in inducing astrocyte NO production, anti-inflammatory cytokines have the capacity to downregulate NO production by IL-1-stimulated astrocytes. © 1995 Wiley-Liss, Inc.     

1,25(OH)_2D_3减轻小鼠局灶性脑缺血再灌注后炎性反应

目的探讨1,25(OH)_2D_3对小鼠局灶性脑缺血再灌注后炎性反应的作用及其机制。方法造模前,通过一个月低维生素D饮食喂养,小鼠随机分为假手术组、局部缺血再灌注组(模型组)和1,25(OH)_2D_3组(治疗组)。造模前3 d始,假手术组和模型组每天腹腔注射2.4%乙醇,治疗组腹腔注射1,25(OH)_2D_3,共持续6 d。再灌注72 h后,Zea Longa法对鼠进行神经功能评分,干湿重法测量缺血侧脑组织含水量,RT-PCR法检测缺血侧半球IL-1βmRNA和TNF-αmRNA表达,采用Western blot法检测缺血侧半球NF-κB p65和Claudin-5的表达。结果与模型组比较,缺血再灌注后72 h,治疗组小鼠神经功能评分较低,缺血侧半球脑含水量、IL-1βmRNA、TNF-αmRNA和NF-κB p65表达显著减少,Claudin-5表达显著增加,差异均有统计学意义(P0.05)。结论 1,25(OH)_2D_3减轻小鼠局灶性脑缺血再灌注损伤后炎性反应,其机制通过抑制NF-κB的活化有关。     

IL‐1β enhances the antibacterial activity of astrocytes by activation of NF‐κB

Astrocytes have important immune functions in CNS, and astrocytes stimulated by interferon‐γ were showed to have direct antimicrobial function. However whether astrocytes without the stimulation of cytokines have antibacterial function, and how this function is regulated are still largely unknown. In this study, we found that primary cultured astrocytes inhibited the growth of both gram‐negative and gram‐positive bacteria. Further more, we showed that interleukin‐1β (IL‐1β) enhanced the antibacterial effect in a dose‐dependent manner, and the antibacterial effect of astrocytes from IL‐1β receptor‐deficient mice failed to be enhanced by IL‐1β. IL‐1β stimulated IκBα degradation, NF‐κB nuclear translocation, and transactivation in astrocytes. NF‐κB inhibitors blocked NF‐κB activation and the enhanced antibacterial effect induced by IL‐1β. In addition, overexpression of dominant negative IκBα in astrocytes inhibited IκBα degradation and NF‐κB transactivation, and also inhibited the enhanced antibacterial effect induced by IL‐1β. All these data demonstrated that IL‐1β enhanced the antibacterial activity of astrocytes by activation of NF‐κB. © 2009 Wiley‐Liss, Inc.     

Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Neuron-specific effects of interleukin-1β are mediated by a novel isoform of the IL-1 receptor accessory protein

In the CNS, interleukin-1β (IL-1β) is synthesized and released during injury, infection, and disease, mediating inflammatory responses. However, IL-1β is also present in the brain under physiological conditions, and can influence hippocampal neuronal function. Several cell-specific IL-1-mediated signaling pathways and functions have been identified in neurons and astrocytes, but their mechanisms have not been fully defined. In astrocytes, IL-1β induced both the p38 MAPK and NF-κB (nuclear factor κB) pathways regulating inflammatory responses, however in hippocampal neurons IL-1β activated p38 but not NF-κB. Additionally, IL-1β induced Src phosphorylation at 0.01 ng/ml in hippocampal neurons, a dose 1000-fold lower than that used to stimulate inflammatory responses. IL-1 signaling requires the type 1 IL-1 receptor and the IL-1 receptor accessory protein (IL-1RAcP) as a receptor partner. We previously reported a novel isoform of the IL-1RAcP, IL-1RAcPb, found exclusively in CNS neurons. In this study, we demonstrate that AcPb specifically mediates IL-1β activation of p-Src and potentiation of NMDA-induced calcium influx in mouse hippocampal neurons in a dose-dependent manner. Mice lacking the AcPb, but retaining the AcP, isoform were deficient in IL-1β regulation of p-Src in neurons. AcPb also played a modulatory role in the activation of p38 MAPK, but had no effect on NF-κB signaling. The restricted expression of AcPb in CNS neurons, therefore, governs specific neuronal signaling and functional responses to IL-1β.     

Astrocyte reactivity to unconjugated bilirubin requires TNF-α and IL-1β receptor signaling pathways

Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1β signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNF-α receptor (TNFR)1 and IL-1β receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1β cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

The adhesion molecule and cytokine profile of multiple sclerosis lesions

The expression of the adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and their respective receptors on leukocytes, very late activation antigen-4 (VLA-4) and lymphocyte function–associated antigen-1 (LFA-1), together with a selection of proinflammatory and immunomodulatory cytokines (interleukin [IL]-1, IL-2, IL-4, IL-10, tumor necrosis factor-α [TNF-α], transforming growth factor-β [TGF-β], and interferon-γ [IFN-γ]) was examined by immunocytochemistry in multiple sclerosis (MS) lesions of different ages and compared with central nervous system (CNS) tissue from other neurological diseases, both inflammatory and noninflammatory, and normal CNS tissue. These molecules play key roles in lymphocytic infiltration and interactions during tissue inflammation and are in large part normally not expressed by CNS cells. High levels of expression of all the molecules tested were found in MS, particularly in chronic active lesions. Positivity for all molecules was also seen in other neurological diseases, even in noninflammatory conditions. There was some suggestion that the VCAM-1/VLA-4 adhesion pathway was expressed at higher levels in chronic MS lesions, while ICAM-1/LFA-1 was used more uniformly in lesions of all ages. Of the cytokines examined, there was increased expression of TNF-α and IL-4 in MS; this was found to be statistically significant when compared with noninflammatory neurological diseases. The expression of most adhesion molecules and some cytokines was negligible in normal CNS tissue although low-level reactivity for ICAM-1 TGF-β, IL-4, TNF-α, and IL-10 was detected, perhaps indicative of immunoregulatory mechanisms. Microglial cells and astrocytes were the major CNS cell types expressing cytokines. The results indicate a potential in the CNS for widespread induced expression of molecules involved in the inflammatory cascade. No adhesion or cytokine molecule or pattern of expression unusual for MS was apparent.     

Multiple sclerosis: myelin basic protein induced mRNA expression of proinflammatory cytokines in mononuclear cells is suppressed by interferon-β 1b in vitro

Beta-interferon (IFN-β) is a promising treatment in multiple sclerosis (MS), reducing the exacerbation rate and MRI lesion burden, as well as the disease progression in relapsing-remitting MS. IFN-β was originally defined by its antiviral effects, but the interest has recently been focused on its immunomodulatory properties. Myelin basic protein (MBP) is one of several autoantigens considered to be the target for autoaggressive immune responses, which eventually might lead to the development of MS. To study in-vitro effects of IFN-β1b on MBP induced cytokine expression, mRNA for the Th1 cytokines IFN-γ and TNF-α, the Th2 related IL-4 and IL-6, the cytolytic perforin and the immune response downregulating TGF-β was measured with in situ hybridization after culture of blood mononuclear cells (MNC) in the presence and absence of MBP. Numbers of cells expressing IFN-γ, TNF-α, perforin and IL-4 mRNA were significantly suppressed after culture with 10 U/ml IFN-β1b. No such effect was seen on MBP induced IL-6 or TGF-β mRNA expression. These observations suggest that one of the major effects of IFN-β1b is the induction of a shift in the cytokine mRNA profile towards a more immunosuppressive pattern. In parallel in vitro tests, the control substance dexametasone (40 μg/ml) reduced the numbers of cells expressing mRNA for all cytokines under study with the exception of TGF-β, to an extent equal to or even more pronounced than IFN-β1b.     

Inhibition of nuclear factor-kappa B sensitises anterior pituitary cells to tumour necrosis factor-α- and lipopolysaccharide-induced apoptosis

Nuclear factor-kappa B (NF-κB), an important pro-inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α activate NF-κB signalling. Oestrogens were shown to suppress NF-κB activation. Oestrogens exert a sensitising action to pro-apoptotic stimuli such as LPS and TNF-α in anterior pituitary cells. In the present study, we show by western blotting that 17β-oestradiol (E(2)) decreases TNF-α-induced NF-κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E(2) decreases LPS-induced NF-κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF-κB pathway sensitises anterior pituitary cells to pro-apoptotic stimuli, we used an inhibitor of NF-κB activity, BAY 11-7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF-α-induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF-κB signalling enhances E(2)-sensitising effect to TNF-α-induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS-induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS-induced expression of Bcl-xL in pituitaries of OVX rats is decreased by E(2) administration. Our results show that inhibition of the NF-κB signalling pathway sensitises anterior pituitary cells to the pro-apoptotic action of LPS and TNF-α. Because E(2) inhibits LPS- and TNF-α-activated NF-κB nuclear translocation, the present study suggests that E(2) sensitises anterior pituitary cells to TNF-α- and LPS-induced apoptosis by inhibiting NF-κB activity.     

银杏总黄酮对脂多糖诱导的小胶质细胞炎症反应的抑制作用

目的探讨银杏总黄酮(TFG)对脂多糖(LPS)诱导的小胶质细胞(MG)炎症反应的抑制作用。方法通过LPS诱导小胶质细胞系(BV-2)小胶质细胞活化建立神经系统炎症模型,分别用不同浓度TFG(0、40、80、120和160 mg/m L)处理细胞,CCK-8方法检测各组细胞活性,选取TFG(80 mg/m L)预处理细胞,倒置相差显微镜观察小胶质细胞形态变化。ELISA法检测白细胞介素-1β(IL-1β)、白细胞介素6 (IL-6)和肿瘤坏死因子-α(TNF-α)水平,Western blot检测Toll样受体4(TLR4)、抑制性卡巴蛋白(IκB-α)、核因子κB(NF-κB)/p65蛋白表达水平。结果低剂量组TFG(40和80 mg/m L)对小胶质细胞活性无明显影响,差异无统计学意义(P 0. 05);高剂量的TFG(120和160 mg/m L)显著抑制小胶质细胞活性,差异具有统计学意义(P 0. 05)。与LPS组相比,TFG+LPS组显著抑制LPS诱导的小胶质细胞形态变化及细胞炎症因子IL-1β、IL-6、TNF-α表达和释放(P 0. 05);明显降低TLR4蛋白表达水平(P 0. 05);显著增加胞质内IκB-α的表达(P 0. 05);抑制NF-κB/p65向核内转移(P 0. 05)。结论 TFG抑制LPS所诱导的小胶质细胞炎症反应效果良好,对以神经炎症为病理特征的神经退行性病变有一定治疗作用。     

Trichostatin A enhances OGD-astrocyte viability by inhibiting inflammatory reaction mediated by NF-κB

In this study we investigate the protective effects of Trichostatin A (TSA) on astrocyte injury after oxygen–glucose deprivation (OGD) and further explore its possible protective mechanisms of inhibiting inflammatory reaction mediated by nuclear factor-κB (NF-κB). In the in vitro model of astrocyte OGD, TSA treatment was used at different doses and time points before deprivation. Astroglial viability was determined by MTT assay. Then tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 mRNA were measured by RT-PCR. Furthermore, the expression of phosphorylated p65, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK), MAPK/c-Jun N-terminal (JNK) and MAPK/p38 was assayed by Western blot. The results showed that TSA at the five doses (12.5, 25, 50, 100, and 200 ng/ml) significantly enhanced the astrocytes viability by 25.3%, 46.1%, 37.5%, 34.9%, and 22% of the vehicle, respectively. The level of TNF-α, IL-1β and IL-6 mRNA in astrocytes was increased after OGD and down-regulated by TSA (p < 0.05). In addition, the phosphorylation p65 was markedly activated in the astrocytes after OGD compared to the control (p < 0.05). TSA inhibited phosphorylation of p65 but did not affect the MAPK pathway. Our results suggest that TSA protects astrocytes from damage after OGD by the inhibition of the inflammatory reaction and this protection is at least partially through the suppression of phosphorylation of NF-κB p65.