Down-regulation of beta2-adrenergic receptor expression by exercise training increases IL-12 production by macrophages following LPS stimulation

Three-week exercise training decreased the steady state level of beta(2)-adrenergic receptor (beta(2)AR) mRNA in peritoneal macrophages from BALB/c mice. When peritoneal macrophages from both exercise-trained and sedentary control mice were stimulated with lipopolysaccharide (LPS), interleukin (IL)-12 mRNA and protein expression was markedly higher in trained mice than in control mice. To determine whether enhanced production of IL-12 was associated with decreased expression of beta(2)AR, we transfected the macrophage cell line, RAW264, with a eukaryotic expression vector containing beta(2)ar cDNA, establishing a cell line overexpressing beta(2)AR (RAWar). Following LPS stimulation, IL-12 mRNA and protein expression was significantly lower in RAWar cells than in RAW264 cells transfected with vector alone (RAWvec). Furthermore, when the expression of transfected beta(2)AR in RAWar cells was down-regulated by a tetracycline repressor-regulated mammalian expression system, expression of IL-12 mRNA and protein following LPS stimulation tended to return to the levels in RAWvec cells. These findings indicate that macrophage production of IL-12 following LPS stimulation is regulated by the expression level of beta(2)AR, suggesting that the down-regulation of beta(2)AR expression associated with exercise training improves IL-12-induced type 1 helper T cell-mediated immune responses.     

表没食子儿茶素-3-没食子酸酯抑制脂多糖诱导的巨噬细胞促炎因子TNF-α和IL-1β基因表达

表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)具有抗氧化、抗癌、抗炎等多种生物学特性,但对巨噬细胞中表达TNF-α及IL-1β的报告尚存在争议.本文旨在探索EGCG对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞和RAW264.7细胞促炎细胞因子Tnf-α和Il-1β基因表达的影响.MTT结果显示,0~100μmol/L EGCG对RAW264.7细胞活力没有影响;实时荧光定量PCR(qRT-PCR)和ELISA分析显示,1 mg/L LPS可显著升高小鼠腹腔巨噬细胞和RAW264.7细胞Tnf-α和Il-1βmRNA和蛋白水平,EGCG单独处理对巨噬细胞Tnf-α和Il-1β的基因表达与蛋白生成没有影响,但可以抑制LPS诱导的巨噬细胞Tnf-α和Il-1β的基因表达与蛋白生成,并存在剂量依赖效应.上述结果提示,EGCG可以剂量依赖方式抑制LPS诱导的巨噬细胞促炎细胞因子Tnf-α和Il-1β的表达,这可能与EGCG的抗炎效应有关.     

Kruppel样因子4对内毒素所致IL-6基因表达的调控及机制研究

探讨Kruppel样因子4(KLF4)对内毒素所致白介素(IL-6)的基因表达以及释放的影响,并对其调控机制做了初步研究．使用RT-PCR和Western blot检测KLF4 mRNA和蛋白质的表达．采用KLF4过表达的RAW264.7巨噬细胞株或反义寡核苷酸技术抑制内源性KLF4的表达,用RT-PCR和ELISA检测内毒素(LPS)刺激后IL-6 mRNA和蛋白质的表达．采用荧光素酶报告基因检测RAW264.7细胞中KLF4过表达对IL-6基因启动子报告基因转录活性的影响．使用EMSA法检测细胞中KLF4与IL-6基因启动子区KLF4元件的结合．结果表明：LPS可以诱导RAW264.7巨噬细胞KLF4的表达以及IL-6蛋白表达．KLF4过表达明显抑制IL-6的mRNA和蛋白质的表达,而KLF4缺失使这种作用消失．荧光素酶报告基因的结果显示,KLF4可以抑制LPS所致的IL-6基因启动子的转录活性．EMSA显示KLF4不能与IL-6启动子区的KLF4结合元件直接结合．结果表明,LPS可以促进RAW264.7小鼠巨噬细胞KLF4的表达和IL-6的释放．KLF4能抑制LPS诱导的IL-6表达和释放,其机制是抑制IL-6启动子的转录活性,但KLF4的抑制作用不是通过直接与IL-6基因的启动子区相结合而实现的．     

Control of Early Theiler's Murine Encephalomyelitis Virus Replication in Macrophages by Interleukin-6 Occurs in Conjunction with STAT1 Activation and Nitric Oxide Production

During Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages, it is thought that high interleukin-6 (IL-6) levels contribute to the demyelinating disease found in chronically infected SJL/J mice but absent in B10.S mice capable of clearing the infection. Therefore, IL-6 expression was measured in TMEV-susceptible SJL/J and TMEV-resistant B10.S macrophages during their infection with TMEV DA strain or responses to lipopolysaccharide (LPS) or poly(I · C). Unexpectedly, IL-6 production was greater in B10.S macrophages than SJL/J macrophages during the first 24 h after stimulation with TMEV, LPS, or poly(I · C). Further experiments showed that in B10.S, SJL/J, and RAW264.7 macrophage cells, IL-6 expression was dependent on extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) and enhanced by exogenous IL-12. In SJL/J and RAW264.7 macrophages, exogenous IL-6 resulted in decreased TMEV replication, earlier activation of STAT1 and STAT3, production of nitric oxide, and earlier upregulation of several antiviral genes downstream of STAT1. However, neither inhibition of IL-6-induced nitric oxide nor knockdown of STAT1 diminished the early antiviral effect of exogenous IL-6. In addition, neutralization of endogenous IL-6 from SJL/J macrophages with Fab antibodies did not exacerbate early TMEV infection. Therefore, endogenous IL-6 expression after TMEV infection is dependent on ERK MAPK, enhanced by IL-12, but too slow to decrease viral replication during early infection. In contrast, exogenous IL-6 enhances macrophage control of TMEV infection through preemptive antiviral nitric oxide production and antiviral STAT1 activation. These results indicate that immediate-early production of IL-6 could protect macrophages from TMEV infection.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Enhancement of phagocytosis and cytotoxicity in macrophages by tumor-derived IL-18 stimulation

Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b⁺ cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells. [BMB Reports 2014; 47(5): 286-291]     

Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411.

CD14-transfected Chinese hamster ovary K1 fibroblasts (CHO/CD14) respond to lipopolysaccharide (LPS) by metabolizing arachidonic acid and with translocation of NF-kappaB to the nucleus. Although previous experiments failed to identify the production of tumor necrosis factor-alpha and interleukin (IL)-1beta by CHO/CD14 cells, LPS did induce the expression of IL-6 mRNA and the subsequent release of the IL-6 protein. To identify additional LPS-inducible genes, a cDNA library derived from LPS-stimulated CHO/CD14 cells was screened by subtractive hybridization. Fourteen genes were found to be expressed differentially, and two were analyzed in detail: hop (Hsp70/Hsp90-organizing protein), which is the hamster homologue of the stress-inducible yeast gene, STI1, and clone H411, which encodes a novel LPS-inducible growth factor. In response to LPS, the expression of Hop mRNA was also increased in both the murine macrophage cell line, RAW 264.7, as well as in primary hamster macrophages. This suggested that the up-regulation of Hop expression is part of the macrophage stress response to LPS. Clone H411 encodes a protein in the epidermal growth factor-like repeat protein family. Overexpression of H411 cDNA in the RAW 264.7 macrophage cell line promoted an increased growth rate, suggesting that expression of H411 is part of the proliferative cell response to LPS. Both Hop and H411 represent novel gene products not previously recognized as part of the complex biological response to endotoxin.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.     

γ-Tocotrienol inhibits lipopolysaccharide-induced interlukin-6 and granulocyte colony-stimulating factor by suppressing C/EBPβ and NF-κB in macrophages

Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) β appears to be involved in IL-6 formation because LPS induces C/EBPβ up-regulation, which parallels IL-6 production, and knockdown of C/EBPβ with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPβ and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPβ without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPβ target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBβ and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.     

2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing IFN-beta production

2-aminopurine (2-AP) is widely used as a specific inhibitor for double stranded-RNA dependent protein kinase (PKR). Here we report that 2-AP can inhibit lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production through the prevention of interferon (IFN)-beta production. 2-AP significantly inhibited NO production in LPS-stimulated RAW 264 murine macrophage cells. 2-AP also reduced the expression of IFN-beta and IFN-inducible genes, such as IFN-gamma-inducible protein (IP)-10 and immune-responsive gene (IRG)-1, and the inducible type of NO synthase (iNOS) mRNA in response to LPS. The addition of exogenous IFN-beta restored 2-AP-inhibited NO production in response to LPS. On the other hand, there was only partial inhibition by 2-AP of nuclear factor (NF)-kappaB activation, IL-6 mRNA expression and tumor necrosis factor (TNF)-alpha production. These results suggested that 2-AP inhibited LPS-induced IFN-beta production by preventing Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling rather than myeloid differentiation factor (MyD) 88-dependent signaling, resulting in the inhibition of NO production.