Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2–induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.     

ANX A2在抗磷脂抗体诱导THP-1细胞组织因子表达中的作用

目的： 构建针对人膜联蛋白A2（ANX A2）的RNA干扰（RNAi）慢病毒表达载体,转染THP-1细胞,探讨ANX A2在抗磷脂抗体（APL）诱导单核细胞组织因子（TF）表达中的作用。方法：设计4条ANX A2特异性RNAi的寡核苷酸序列,与慢病毒载体pGCSIL-GFP连接,PCR及测序鉴定正确后,Western蛋白印迹法筛选出有效干扰序列。包装293T细胞获得重组慢病毒LV-RNAi-ANX A2,感染单核细胞株THP-1,观察细胞ANX A2 mRNA和蛋白表达下降程度。再用APL/β2GPI复合物刺激干扰后的THP-1细胞,观察TF mRNA表达及TF活性变化。结果：成功构建RNAi慢病毒载体并筛选出有效干扰片段;包装293T细胞后病毒滴度为3×1012TU/L;感染THP-1后细胞ANX A2 mRNA和蛋白均被沉默。APL/β2GPI复合物刺激干扰后的THP-1细胞,其TF表达下降至基础水平。结论：构建的慢病毒表达载体能显著抑制THP-1细胞ANX A2的表达,进而影响APL诱导的TF表达,证明ANX A2在APL诱导的单核细胞表达TF过程中具有重要作用。     

The A2aR adenosine receptor controls cytokine production in iNKT cells

The purine nucleoside adenosine is an important anti‐inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor‐mediated mechanisms. Invariant NKT (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN‐γ production by iNKT cells. We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR and A3R on mouse iNKT cells. We show that IL‐4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL‐4. These data are supported by A2aR‐deficient mice, which exhibit largely decreased levels of IL‐4, IL‐10 and TGF‐β concomitantly with an increase of IFN‐γ upon α‐galactosylceramide administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL‐4 and IL‐10 by iNKT cells. These data suggest adenosine:A2aR‐mediated mechanisms can control the cytokine secretion pattern of iNKT cells.     

Oxidative microenvironment exerts an opposite regulatory effect on cytokine production by Th1 and Th2 cells

Oxidative stress occurs in allergic disorders and immunologic inflammatory responses and reactive oxygen metabolites have an additional role of cell-signaling mediators, influencing many biological processes. Using in vitro derived Th1 and Th2 clones or T cells derived from autoimmune thyroiditis we study the ability of Th1 or Th2 cells to expand and produce cytokine in an oxidative environment. We found that low-doses of H2O2 reduce the INF-gamma production of activated Th1 clones and potentiate the IL-4 secretion of activated Th2 clones. These effects were not due to altered cell proliferation and are not transient, since the modified secretion profile was still retained after 1 week from H2O2 stimulation by both Th1 and Th2 cells. H2O2 influence the profile of cytokine secretion in both Th1 and Th2. These effects are long lasting and are the result of an action of H2O2 on T cell. In conclusion we demonstrate that oxidative stress plays an important role in the pathogenesis of allergic respiratory diseases and can up-regulate Th2-driven inflammation, thus contributing to increase disease severity, bronchial hyper-responsiveness and airway remodeling.     

Role of potassium channels in the regulation of cytokine release from THP-1 cells

The role of K⁺ channels in the regulation of cytokine release was investigated. Quinine, a non-selective K⁺ channel blocking agent inhibited both TNF and IL-1 release but had non-specific effects on cell function. Glipizide and glibenclamide, inhibitors of ATP-sensitive K⁺ channels, reduced IL-1 release but not TNF, whereas apamin, a selective inhibitor of low-conductance Ca²⁺-sensitive channels, inhibited TNF release, potentiated IL-1 but had no effect on IL-6 or IL-8 release. These results suggest that K⁺ channels may differentially regulate cytokine production by THP-1 cells.     

Role of potassium channels in the regulation of cytokine release from THP-1 cells

The role of K⁺ channels in the regulation of cytokine release was investigated. Quinine, a non-selective K⁺ channel blocking agent inhibited both TNFα and IL-1β release but had non-specific effects on cell function. Glipizide and glibenclamide, inhibitors of ATP-sensitive K⁺ channels, reduced IL-1β release but not TNFα, whereas apamin, a selective inhibitor of low-conductance Ca²⁺-sensitive channels, inhibited TNFα release, potentiated IL-1β but had no effect on IL-6 or IL-8 release. These results suggest that K⁺ channels may differentially regulate cytokine production by THP-1 cells.     

Molecular pharmacology of the beta-adrenergic receptor on THP-1 cells

The beta-adrenergic receptor, its occupancy and subsequent modulation of intracellular cAMP, and mRNA expression were characterized for the promonocytic leukemia cell line THP-1. We report that THP-1 cells appear to express a beta-1 receptor with a K_d of 1.8 ± 0.3 × 10⁻¹¹μM and a B max of 108 ± 0.07 fmole/mg protein using ¹²⁵I-iodocyanopindolol (¹²⁵I-ICYP). The potency of various beta-adrenergic agonists to compete for the ¹²⁵I-ICYP binding site followed the order: isoproterenol (0.8 μM)>dobutamine (2.1 μM)>salbutamol (3 μM)>epinephrine (3.8 μM)>soterenol) (4.6 μM)>terbutaline (11.1 μM)>norepinephrine (13.8 μM). Occupancy of the beta receptor on THP-1 cells results in activation of adenyl cyclase suggesting that these cells have a functional beta-adrenergic receptor. This receptor also has specific immunoregulatory properties, reducing message levels for tumor necrosis factor — but not interleukin 1, following treatment with isoproterenol (approximate EC-50 of 0.01 μM). We conclude, based on the above criteria, that THP-1 cells express a beta-1 receptor which, following ligand binding, results in increased cAMP leading to downregulation of TNF expression.     

miR-146a对THP-1细胞靶基因AUF1调控及细胞因子表达的影响

目的探讨miR-146a对人急性单核白血病细胞系(THP-1)靶基因AU碱基富集区结合降解因子1(AUF1)表达的调控及细胞因子表达的影响。方法构建miR-146a过表达及抑制表达慢病毒载体并转染THP-1细胞,以正常培养的THP-1细胞为对照。用实时定量PCR法检测THP-1细胞AUF1 mRNA表达;蛋白质印迹法检测THP-1细胞AUF1蛋白的表达;酶联免疫吸附试验检测THP-1细胞培养基上清液白细胞介素-8(IL-8)及白细胞介素-35(IL-35)浓度。结果过表达miR-146a,可导致THP-1细胞AUF1 mRNA及蛋白表达下调(P0.01,P0.05);THP-1细胞上清液IL-8及IL-35浓度降低(P0.01,P0.05)。抑制miR-146a表达,导致THP-1细胞上清液IL-8及IL-35浓度明显增高(P0.01,P0.01)。结论 AUF1是miR-146a的靶基因。miR-146a可以调控THP-1细胞上清液IL-8、IL-35浓度。IL-8的改变引起相应IL-35的改变,一起参与炎性反应。     

IM-133N modulates cytokine secretion by RAW264.7 and THP-1 cells

An indigenous herbal extract IM-133N containing extracts of Prosopis glandulosa Torr and Symplocos racemosa Roxb were evaluated for potential immunomodulatory effects using RAW264.7 and THP-1 cells. The incubation of the cells for 24?h with IM-133N over a dose range 0–125?µg/ml did not cause cytotoxicity that exceeded 10%. The results indicated that non-cytotoxic doses of IM-133N effectively up-regulated iNOS, TNFα, IL-6, IL-10, IL-8 and IFNγ gene expression in both the RAW264.7 and THP-1 cells. The results also indicated IM-133N elicited dose-related increases in nitric oxide (NO) and tumor necrosis factor (TNF)-α production by RAW264.7 or THP-1 cells. These results demonstrated that IM-133N could stimulate NO and induced pro-inflammatory cytokine expression by monocytes/macrophages. As clinical studies have shown IM-133N to be an effective immunomodulator without any adverse effects, the results of the present study provide further support for the potential use of this agent as an immunostimulant or as an immunotherapy adjuvant.     

Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells.

Neutrophil-dependent inflammation dependent on monosodium urate (MSU) crystal-induced IL-8 expression occurs in gout. MSU crystals activate phagocyte Src family tyrosine kinases and the serine/threonine kinase p70s6k. Thus, using monocytic THP-1 cells, we assessed the potential for Src family kinases and p70s6k to mediate MSU-induced IL-8 expression. MSU crystals induced phosphorylation of p70s6k and the Src kinases c-Src, Lyn, Hck, and Fyn. IL-8 expression was attenuated more by the Src kinase inhibitor PP1 than by the p70s6k inhibitor rapamycin. PP1 inhibited crystal-induced phosphorylation of ERK1/2 and IkappaBalpha and suppressed IkappaB kinase (IKK) activation and NF-kappaB binding to the IL-8 promoter, signals that mediate MSU-induced IL-8 expression. Transfection of the native Src inhibitor, C-terminal Src kinase (Csk), also suppressed crystal-induced c-Src, ERK1/2, and IkappaBalpha phosphorylation and IL-8 expression. We conclude that Src family tyrosine kinase signaling plays a significant role in MSU crystal-induced IL-8 expression via stimulation of ERK1/2 pathway and NF-kappaB activation.     

Regulation of cytokine and chemokine expression by the ribotoxic stress response elicited by Shiga toxin type 1 in human macrophage-like THP-1 cells

Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1β (IL-1β) and chemokines IL-8, growth-regulated protein-β, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.     

伤寒杆菌IVB型菌毛激活PKC信号通路诱导THP-1细胞IL-6表达

目的 探讨伤寒杆菌IVB型菌毛激活PKC信号通路诱导THP-1细胞IL-6表达。方法将THP-1细胞分别与有IVB型菌毛的A21-6菌、缺失IVB型菌毛的pilS^-菌共同孵育,分别检测蛋白激酶C(PKC)活性、IL-6产量及IL-6 mRNA的表达水平;用PKC抑制剂DECA预处理THP-1细胞,然后再以A21-6菌诱导,测定PKC活性和IL-6产量。结果THP-1细胞经有IVB型菌毛的A21-6菌刺激后,IL-6的产生水平、mRNA的表达水平以及PKC活性均显著高于相应的处理组。PKC活性在一定范围内呈时效关系,刺激10min后即明显升高,并于30min左右达到峰值。PKC抑制剂DECA一定程度上能够抑制IVB型菌毛诱导的THP-1细胞PKC活性和IL-6产生水平。结论IVB型菌毛在伤寒杆菌诱导单核／巨噬细胞产生IL-6过程中是一种重要的刺激因素;PKC参与了该过程的细胞内信号转导。     

Actinobacillus actinomycetemcomitans Y4 capsular polysaccharide induces IL-1beta mRNA expression through the JNK pathway in differentiated THP-1 cells

Capsular polysaccharide from Actinobacillus actinomycetemcomitans Y4 (Y4 CP) induces bone resorption in a mouse organ culture system and osteoclast formation in mouse bone marrow cultures, as reported in previous studies. We also found that Y4 CP inhibits the release of interleukin (IL)-6 and IL-8 from human gingival fibroblast (HGF). Thus Y4 CP induces various responses in localized tissue and leads to the secretion of several cytokines. However, the effects of Y4 CP on human monocytes/macrophages are still unclear. In this study, THP-1 cells, which are a human monocytic cell line, were stimulated with Y4 CP, and we measured gene expression in inflammatory cytokine and signal transduction pathways. IL-1beta and tumour necrosis factor (TNF)-alpha mRNA were induced from Y4 CP-treated THP-1 cells. IL-1beta mRNA expression was increased according to the dose of Y4 CP, and in a time-dependent manner. IL-1beta mRNA expression induced by Y4 CP (100 microg/ml) was approximately 7- to 10-fold greater than that in the control by real-time PCR analysis. Furthermore, neither PD98059, a specific inhibitor of extracellular signal-regulated kinase nor SB203580, a specific inhibitor of p38 kinase prevented the IL-1beta expression induced by Y4 CP. However, JNK Inhibitor II, a specific inhibitor of c-Jun N-terminal kinase (JNK) prevented the IL-1beta mRNA expression induced by Y4 CP in a concentration-dependent manner. These results indicate that Y4 CP-mediated JNK pathways play an important role in the regulation of IL-1beta mRNA. Therefore, Y4 CP-transduced signals for IL-1beta induction in the antibacterial action of macrophages may provide a therapeutic strategy for periodontitis.     

肺炎支原体膜脂蛋白通过TLR2介导mICAM-1表达的上调

目的　观察肺炎支原体膜脂蛋白 (Mp LAMP)对THP 1 (前单核白血病细胞系 )细胞TLR2 (Toll样受体 )与mICAM 1 (膜结合型细胞间黏附分子 1 )表达的影响 ,探讨膜脂蛋白中的活性成分及二者表达的相互关系。方法　用流式细胞术检测Mp LAMP刺激组、抗人TLR2功能纯化抗体(TL2 .1 )阻断组及消化酶处理组的THP 1细胞mICAM 1和TLR2的表达水平。结果　与对照组相比 ,不同刺激浓度的Mp LAMP对THP 1细胞膜上TLR2及mICAM 1的表达均有明显的上调作用 (P <0 .0 5 ) ,且表达水平的增加对Mp LAMP的刺激具有浓度依耐性 ;用TL2 .1将THP 1细胞膜上的TLR2阻断后 ,Mp LAMP对mICAM 1表达的诱导作用明显受到抑制 (P <0 .0 1 ) ,其阻断效果随TL2 .1量的增加而加强 ;酶消化试验证实Mp LAMP的活性成分是其脂质部分。相关分析表明 ,mICAM 1在THP 1细胞上的表达水平与TLR2密切相关 (r=0 .989,P <0 .0 1 )。结论　肺炎支原体膜脂蛋白通过TLR2介导上调THP 1细胞mICAM 1的表达 ,在一定程度上影响着肺炎支原体感染所导致的炎性反应的强弱。     

The chemokine receptor CCR8 mediates rescue from dexamethasone-induced apoptosis via an ERK-dependent pathway

Several chemokines have been shown to regulate cellular apoptosis following discrete stimuli. It was previously demonstrated that the CC chemokine CCL1 (I-309) rescues thymic lymphoma cells from apoptosis by unknown mechanisms. The aim of our study was to characterize the role of the CC chemokine receptor 8 (CCR8), the only described receptor for CCL1, in the rescue of murine thymic lymphoma cells and murine thymocytes from dexamethasone (dex)-induced apoptosis. We show here that the CCR8-restricted agonist Kaposi sarcoma-associated herpesvirus-encoded chemokine viral macrophage-inflammatory protein-1 (vMIP-1) rescues thymic lymphoma cells from dex-induced apoptosis, similar to CCL1, and that such rescue is extracellular-regulated kinase-dependent. Although it has been hypothesized that the rescuing effect of CCL1 from apoptosis could be CCR8-mediated, here, we formally demonstrate the role of such receptor as its selective antagonist encoded by the MC148 gene of molluscum contagiosum virus MC148/vMCC-I inhibits v-MIP-1- and CCL1-induced rescue activity. In addition, CCR8 ligands inhibit dex-induced apoptosis of murine thymocytes with potential implications for thymic selection.     

The bradykinin B2 receptor mediates hypoxia/reoxygenation induced neuronal cell apoptosis through the ERK1/2 pathway

The bradykinin B2 receptor (B2R) mediates many physiological processes such as hypotension, inflammation and blood-vessel permeability. Hypoxia/reoxygenation (H/R) induces neuronal cell apoptosis. It was found that B2R expression was enhanced in primary cultured cortical neurons after H/R treatment. Addition of bradykinin (BK) alleviated the neuronal damage from H/R. This protective effect of BK was inhibited by the B2R antagonist, HOE140, and the ERK1/2 antagonist, PD98059. The phosphorylation of ERK1/2 was increased under H/R, and the addition of BK enhanced this effect. These results indicate that B2R plays an important role in protecting neurons from damage induced by H/R and this effect may function through the ERK1/2 pathway.