In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4⁺ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.     

An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 M?) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF M?). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 M? and GM-CSF M? exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 M? versus GM-CSF M?, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 M? and GM-CSF M? (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 M? contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways.     

Suppression of inflammation response by a novel A₃ adenosine receptor agonist thio-Cl-IB-MECA through inhibition of Akt and NF-κB signaling

Adenosine, a purine nucleoside, is released from metabolically active cells into extracellular space and plays an important role in various pathophysiological processes. Adenosine regulates many biological responses including inflammation by the interaction with their receptors such as A₁, A_2A, A_2B, and A₃. Especially, A₃ adenosine receptor (A₃AR) is considered to be expressed in macrophage cells. To the end, A₃AR agonists have been reported to have an anti-inflammatory activity. In our continuous efforts to develop new anti-inflammatory agents, we found a novel adenosine analog, 2-chloro-N⁶-(3-iodobenzyl)-4′-thioadenosine-5′-N-methyluronamide (thio-Cl-IB-MECA), was a potent human A₃AR agonist. The study was designed to investigate whether thio-Cl-IB-MECA has an anti-inflammatory potential in mouse macrophage RAW 264.7 cells and mouse sepsis model in vivo. Thio-Cl-IB-MECA exhibited an effective anti-inflammatory activity. The expression of pro-inflammatory biomarkers including inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor (TNF-α) was suppressed by the treatment of thio-Cl-IB-MECA in the protein and mRNA levels in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Further examination revealed that thio-Cl-IB-MECA inhibited LPS-induced phosphatidylinositol 3-kinase (PI3 kinase)/Akt activation, NF-kB binding activity, and β-catenin expression. In addition, in in vivo LPS-induced mouse endotoxemia model, thio-Cl-IB-MECA exerted the increase of survival rate compared to vehicle-treated mouse. The analysis of the protein levels of iNOS, IL-1β, and TNF-α was also suppressed by the compound-treated groups in lung tissues. These results suggest that thio-Cl-IB-MECA might have an anti-inflammatory activity through the inhibition of pro-inflammatory cytokine expression by modulating PI3K/Akt and NF-κB signaling pathways.     

M-CSF对RAW264.7细胞氧化损伤的保护作用

揭示巨噬细胞集落刺激因子是否能保护单核巨噬细胞免受过氧化损伤。方法以Ｌ９２９细胞条件培养液作为Ｍ－ＣＳＦ来源,以细胞显微形态,细胞活力等指标,观察了Ｍ－ＣＳＦ对叔丁基氢化物诱导的巨噬细胞系ＲＡＷ２６４．７细胞氧化损伤的影响。结论Ｍ－ＣＳＦ对单核巨噬细胞的氧化损伤具有保护作用。     

ARIP2在小鼠巨噬细胞系RAW264.7中对IL-1β分泌的抑制作用

研究ARIP2在巨噬细胞中对IL-1β分泌的调节作用,探讨激活素A与巨噬细胞活化的可能关系及其作用机制。为了分析ARIP2在小鼠巨噬细胞系RAW264.7细胞中的表达及其生物学作用,实验采用RT-PCR方法检测RAW264.7细胞中ARIP2 mRNA的表达,ELISA方法观察过表达ARIP2对RAW264.7细胞分泌IL-1β的影响。RAW264.7细胞能够表达特异性ARIP2 mRNA,其RNA表达受激活素A刺激呈剂量依赖性增加。ELISA检测结果显示过表达ARIP2可以抑制RAW264.7细胞分泌IL-1β。上述资料提示ARIP2不仅具有抑制激活素诱导的特异基因转录活性,其自身也具有多种生物学活性,可能在激活素A抑制LPS活化巨噬细胞分泌IL-1方面,发挥关键性信号转导调控作用。     

CD49a promotes T‐cell‐mediated hepatitis by driving T helper 1 cytokine and interleukin‐17 production

It is becoming increasingly clear that the T-cell-mediated immune response is important in many diseases. In this study, we used concanavalin A (Con A) -induced hepatitis to investigate the role of CD49a in the molecular and cellular mechanism of the T-cell-mediated immune response. We found that CD49a^−/− mice had significantly reduced levels of serum alanine aminotransferase and were protected from Con A-induced hepatitis. CD49a deficiency led to decreased production of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) after Con A injection. Furthermore, we found that hepatic CD4⁺ T cells and invariant natural killer T cells up-regulated CD49a expression, along with enhanced activation after Con A injection, leading to production of inflammatory cytokines by these T cells. Blockade of CD49a in vivo ameliorated Con A-induced hepatitis with reduced production of IFN-γ and IL-17A. Hence, CD49a promoted Con A-induced hepatitis through enhancing inflammatory cytokine production (IFN-γ and IL-17A) by CD4⁺ T and invariant natural killer T cells. The protective effect of CD49a blockade antibody suggested a new target therapeutic molecule for intervention of T-cell-mediated liver injury.     

CCL3/MIP-1alpha is pro-inflammatory in murine T cell-mediated hepatitis by recruiting CCR1-expressing CD4(+) T cells to the liver

T cell-mediated hepatitis is associated with significant morbidity and mortality worldwide. Levels of C-C chemokine ligand 3/macrophage inflammatory protein-1alpha (CCL3/MIP-1alpha) are elevated in the serum of patients with T cell-mediated liver diseases, but its role is not fully understood. Con A-induced hepatitis is a murine liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of IFN-gamma. In this study, we have used CCL3/MIP-1alpha gene-deficient mice to examine the role of CCL3/MIP-1alpha in the pathogenesis of Con A-induced hepatitis. We demonstrate a novel pro-inflammatory role for CCL3/MIP-1alpha since CCL3/MIP-1alpha deficiency significantly attenuated hepatic injury, both biochemically and histologically. Moreover, the recruitment of CCR1-expressing CD4(+) T cells to the liver after Con A treatment was strikingly attenuated by CCL3/MIP-1alpha deficiency. Correspondingly, hepatic IFN-gamma produced by the recruited CD4(+) T cells was significantly reduced by CCL3/MIP-1alpha deficiency during Con A-induced hepatitis. Furthermore, treatment of mice with a dual CCR1/CCR5 peptide antagonist, methionylated RANTES, also markedly reduced hepatic injury and decreased the numbers of CD4(+) T cells within the liver producing IFN-gamma during Con A-induced hepatitis. These findings demonstrate that blockade of the CCL3/MIP-1alpha-CCR1 pathway may represent a novel therapeutic target for treating T cell-mediated liver diseases.     

微弧氧化镁合金对巨噬细胞肿瘤坏死因子和白细胞介素6的影响

背景：微弧氧化处理可提高镁合金的抗腐蚀性能,延缓其降解速率。 目的：观察微弧氧化处理镁合金对小鼠巨噬细胞肿瘤坏死因子α和白细胞介素6的影响。 方法：在小鼠RAW264.7细胞中分别加入镁合金浸提液(对照组)、微弧氧化处理镁合金浸提液(实验组)及RPMI-1640(空白对照组),1 h后加入脂多糖,作用24 h后,收集细胞上清液,检测肿瘤坏死因子α和白细胞介素6水平,MTT法测定脂多糖刺激前后的RAW264.7细胞活性。 结果与结论：①脂多糖刺激前的RAW264.7细胞活性：对照组>实验组>空白对照组(P均< 0.05)。②脂多糖刺激后的RAW264.7细胞活性：实验组与对照组高于刺激前,但差异无显著性意义;空白对照组明显高于刺激前(P < 0.01)。3组间RAW264.7细胞活性差异无显著性意义(P > 0.05)。③脂多糖刺激后RAW264.7细胞白细胞介素6与肿瘤坏死因子α的表达量：对照组白细胞介素6表达量明显高于实验组和空白对照组(P < 0.05);3组肿瘤坏死因子α表达量两两之间比较差异无显著性意义(P > 0.05)。表明微弧氧化处理镁合金对小鼠巨噬细胞活性无明显影响,同时降低了炎性反应。     

Development and characterisation of monoclonal antibodies reactive with porcine CSF1R (CD115)

Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system. CSF1, alongside a second ligand, interleukin-34 (IL-34), acts by binding to a cell surface receptor (CSF1R). We previously cloned and expressed pig CSF1 and IL-34. Here we produced a pig CSF1R-Ig + pFUSE Fc fusion protein and used it as an immunogen to produce three monoclonal antibodies (ROS8G11, ROS3A5 and ROS3B10) targeted against porcine CSF1R. Specific binding of each monoclonal antibody was confirmed by ELISA, Western blot, flow cytometry and immunocytochemistry. The antibodies did not block CSF1 signalling. The surface expression of CSF1R in pig peripheral blood was restricted to CD14-positive monocytes and was also detected on lung macrophages. These antibodies provided an opportunity to investigate the increase of available CSF1R during pig BMDM differentiation. The new monoclonal antibodies provide useful reagents to support the study of monocyte and macrophage biology in the pig.     

Secretion of colony stimulating factor-1 by human first trimester placental and decidual cell populations and the effect of this cytokine on trophoblast thymidine uptake in vitro

The present in-vitro study using an enzyme-linked immunosorbentassay has identified the cell types responsible for colony stimulatingfactor-1 (CSF-1) production at the first trimester human placentaluterine interface. The major sources were observed to be decidualstromal cells and decidual CD56⁺ natural killer (NK) cells,but decidual CD3⁺ T cells did not produce CSF-1, reflectingfunctional differences between these two decidual lymphoid populations.Of a variety of cytokines tested, only interleukin-2 (IL-2)was found to augment CSF-1 secretion by decidual NK cells. Trophoblastcells also secreted CSF-1, but the amounts were small relativeto decidual stromal cells and NK cells. Therefore, most of theCSF-1 present at the implantation site appears to be maternallyderived. Co-culture of decidual NK cells on a monolayer of irradiatedtrophoblast did not augment CSF-1 secretion by decidual NK cells,indicating that the production of this cytokine is not stimulatedby contact with fetal trophoblast. CSF-1 was found to increase[³H]thymidine uptake by trophoblast cultured on laminin for72 h, but no such response was seen in trophoblast culturedon fibronectin, indicating that these extracellular matrix proteinshave differential effects on the response of trophoblast tothis cytokine.     

IL-34, the rationale for its expression in physiological and pathological conditions

IL-34 is a cytokine that shares one of its receptors with CSF-1. It has long been thought that CSF-1 receptor (CSF-1R) receives signals only from CSF-1, but the identification of IL-34 reversed this stereotype. Regardless of low structural homology, IL-34 and CSF-1 emanate similar downstream signaling through binding to CSF-1R and provoke similar but different physiological events afterward. In addition to CSF-1R, protein-tyrosine phosphatase (PTP)-ζ and Syndecan-1 were also identified as IL-34 receptors and shown to be at play. Although IL-34 expression is limited to particular tissues in physiological conditions, previous studies have revealed that it is upregulated in several diseases. In cancer, IL-34 is produced by several types of tumor cells and contributes to therapy resistance and disease progression. A recent study has demonstrated that tumor cell-derived IL-34 abrogates immunotherapy efficacy through myeloid cell remodeling. On the other hand, IL-34 expression is downregulated in some brain and dermal disorders. Despite accumulating insights, our understanding of IL-34 may not be even close to its nature. This review aims to comprehensively describe the physiological and pathological roles of IL-34 based on its similarity and differences to CSF-1 and discuss the rationale for its disease-dependent expression pattern.     

Effect of macrophage colony stimulating factor overexpression on oxidative injury/resistance of RAW264.7 cells

Oxidative injury to monocytes/macrophages is considered one of the key factors in atherogenesis. Macrophage colony stimulating factor (M-CSF) also plays an important role in the stages of atherosclerosis. Some researchers showed that M-CSF accelerated pathological changes in the early stages of atherosclerosis. However, other reports suggested that exogenous M-CSF could prevent the progress of atherosclerosis. To further investigate the role of M-CSF in atherogenesis and to elucidate the effect of M-CSF on the oxidative injury to monocytes/macrophages, RAW264.7 cell lines overexpressing M-CSF were established by applying the lipofectin transfection method. The oxidative injurious effect of tert-butylhydroperoxide on the established cell lines was investigated. Two M-CSF-transfected RAW264.7 cell lines secreted large amounts of M-CSF. Compared with the non-transfected RAW264.7 cells, M-CSF-overexpressing RAW264.7 cells were more vulnerable to oxidative injury. We conclude that M-CSF could aggravate the oxidative injury due to macrophages in some situations. Received: 21 August 2002 / Accepted: 12 March 2003 Correspondence to Z.-J. Pang     

Involvement of plasma lncRNA GSEC in sepsis discrimination and prognosis,and its correlation with macrophage cell inflammation and proliferation

BackgroundDespite the dysregulation and function of G-quadruplex-forming sequence containing lncRNA (GSEC) have been widely reported in human cancers, there are few available data revealing its role in sepsis.ObjectiveTo assess the expression and function of GSEC in the development of sepsis and its potential molecular mechanism.Materials and methodsA total of 78 sepsis patients, 55 non-sepsis intensive care unit patients, and 42 healthy individuals were enrolled in this study. The expression of GSEC was evaluated in plasma and macrophage cells with polymerase chain reaction. The inflammation response of sepsis patients and macrophage cells was analyzed with an enzyme-linked immunosorbent assay. The diagnostic and prognostic value of GSEC in sepsis patients were estimated by receiver operator curve (ROC) and Cox analysis. The molecular mechanism underlying the function of GSEC was investigated in RAW264.7 cell with luciferase reporter assay and cell transfection.ResultsSignificant upregulation of GSEC was observed in sepsis patients’ plasma, which could discriminate sepsis patients from healthy and non-sepsis individuals. Upregulation of GSEC was positively correlated with inflammation cytokine levels and adverse prognosis of sepsis patients. In vitro, GSEC was found to modulate the expression level of miR-873-3p, which mediated the regulatory effect of GSEC on the inflammation and proliferation of RAW264.7.ConclusionUpregulated GSEC could serve as a biomarker of sepsis pathogenesis and development. GSEC regulates the inflammation and proliferation of macrophage cells through modulating miR-873-3p.     

The effects of inflammatory cytokines on steroidogenic acute regulatory protein expression in macrophages

Objective: To investigate the expression of steroidogenic acute regulatory protein (StAR) in macrophages and the effects of inflammatory cytokines on StAR expression. Methods: The macrophages isolated from ApoE knockout mice and C57BL/6J mice and RAW264.7 cells (a cell line from mouse macrophage. ATCC Number: TIB-71^TM) were cultured in DMEM containing 10% fetal bovine serum. RAW264.7 cells were treated with different inflammatory cytokines (TNF-α, IFN-γ and TGF-β1) and 8-Br-cAMP, a cAMP analog. RT-PCR and Western blot analysis were applied to evaluate the effects of inflammatory cytokines on StAR expression. Results: RT-PCR and Western blot analysis demonstrated the expression of StAR in the macrophages isolated from ApoE knockout mice, C57BL/6J mice and RAW264.7 cells. Proinflammatory cytokines TNF-α and IFN-γ significantly decreased StAR mRNA and protein levels in RAW264.7 cells. The inhibition was dose- and time-dependent. In contrast, anti-inflammatory cytokine TGF-β1 increased StAR mRNA and protein levels. At 1:15 molecular ratio, TGF-β1 blocked the down-regulation of StAR expression mediated by TNF-α. cAMP also induced StAR expression in RAW264.7 cells. When the cells were co-treated with 8-Br-cAMP and TNF-α, 8-Br-cAMP failed to induce StAR expression. Conclusion: Our results provide interesting evidence that inflammatory cytokines regulate StAR expression in macrophages. Received 12 August 2006; returned for revision 28 September 2006; returned for final revision 28 May 2007; accepted by M. Katori 22 June 2007     

Connection between inflammatory processes and transmittor function—Modulatory effects of interleukin-1

Cells in the nervous system can respond to different kinds of stress, e.g. injury, with production and release of inflammatory molecules, including cytokines. One of the most important proinflammatory cytokines is interleukin-1, affecting most organs of the body. The high constitutive expression of interleukin-1 in the adrenal gland provides a source for local and systemic actions, in addition to activated monocytes. In the brain, the constitutive expression is low, but activated microglia produce and release interleukin-1 during pathological conditions such as neurodegenerative disorders (e.g. stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease). Interleukin-1 has an important role in mediating ‘sickness symptoms’ such as fever, in response to infections. Its role in neurodegeneration is not fully elucidated, but there is evidence for involvement in both amyloidosis and tau pathology, major neuropathological hallmarks of Alzheimer's disease. The interleukin-1 family at present consists of 11 members, one of which is the endogenous receptor antagonist. Overexpression of this antagonist in the CNS in a transgenic mouse strain, Tg hsIL-1ra, has allowed studies on morphological and functional effects of blocking interleukin-1 receptor-mediated activity in the brain. Marked alterations of brain morphology such as reduced hippocampal and cortical volume correlate with behavioural deficits. Decreased anxiety and impaired long-term memory are among the consequences. Intact interleukin-1 signalling is important for the brain's ability to adapt to acute and chronic neuroinflammation. Increased amplitude and prolongation of proinflammatory cytokine production underly the behavioural alterations characteristic for ageing. Moreover, deregulated expression of interleukin-1 is associated with ageing-related chronic neurodegenerative disorders.     

The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-β-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.