Single-vesicle tracking reveals the potential correlation of the movement of cell-bound membrane vesicles (CBMVs) with cell migration

The movement of cell-bound membrane vesicles (CBMVs) on migrating cells is poorly understood. We hypothesized that the movement of CBMVs on migrating cells is different from that on non-migrating cells and can be interfered by external stimuli. To test it, single-vesicle tracking was performed to analyze motion type, speed, displacement, and direction of CBMVs on migrating cells treated with different reagents (Ang-1, TNF-α, LPS, VEGFα, endostatin, Cytochalasin D, and nocodazole) among which the former four promoted cell migration whereas the others inhibited cell migration. We found that cell migration changed CBMVs from non-directed to directed motion and that most CBMVs on untreated migrating cells moved along the migration axis. Interestingly, the migration-promoting reagents played positive roles in CBMV movement (improving directed motion, speed and/or maximal displacement, upregulating the amount of vesicles moving in migration direction) whereas the migration-inhibiting reagents played negative roles (impairing/abolishing directed motion, speed and/or maximal displacement, downregulating the vesicles moving forward or causing an even distribution of motion direction). The cytoskeleton (particularly microtubules) probably played vital roles in CBMV movement on migrating cells and mediated the effects of stimuli on vesicle movement. The data may provide important information for understanding the properties, behaviors, and functions of CBMVs.     

Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2

The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW⁴. oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis.     

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.     

Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors

Patients with chronic inflammatory disorders such as rheumatoid arthritis (RA) have a high risk of developing cardiovascular disease. We evaluated the effects of TNF-α and IL-6 on foam cell formation, a pivotal process in atherogenesis. Accumulation of intracellular oxidized LDL (oxLDL) was induced when THP-1/macrophages were stimulated with TNF-α or IL-6. TNF-α induced the expressions of scavenger receptors SR-A and LOX-1, and IL-6 induced SR-A expression. Inhibition of the NF-κB signaling markedly decreased TNF-α-induced foam cell formation and SR-A expression. Serum from RA patients, but not healthy subjects, induced foam cell formation, which was partially reversed by either IL-6 or TNF-α blockade in conjunction with inhibiting the induction of scavenger receptors. The present study clearly showed that in patients with chronic inflammation mediated by TNF-α and IL-6, these cytokines are directly implicated in atherosclerotic plaque formation.     

INTERLEUKIN 6 UPREGULATES TNF-α-DEPENDENT C3-STIMULATING ACTIVITY THROUGH ENHANCEMENT OF TNF-α SPECIFIC BINDING ON RAT LIVER EPITHELIAL CELLS

The authors investigated the role of tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), two major pro-inflammatory cytokines, in the stimulation of the third component of complement (C3) production by rat liver epithelial cells. Though often considered as the most potent inflammatory cytokine, IL-6 alone displayed no activity, whereas TNF-α upregulated C3 production in a dose-dependent manner. However, IL-6 was shown to synergistically stimulate C3 production in the presence of TNF-α. To account for this interaction, it was postulated that IL-6 modulates the binding of TNF-α on liver target cells. That IL-6 increased the binding of TNF-α on the surface of the hepatic cells, whereas TNF-α alone downregulated its own specific binding capacity is reported. Furthermore, this upregulatory effect of IL-6 was mainly attributable to an increase in the number of plasma membrane TNF-α specific receptors, with little change in their affinity. These results suggest that the synergistic IL-6 activity on C3 production may occur, at least partially, through its capacity to upregulate the number of TNF-α receptors on the surface of the rat liver epithelial cells.     

Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-α-induced apoptosis

Tumor necrosis factor-α (TNF-α) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-α-induced apoptosis, we isolated and characterized a novel TNF-α-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-α and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-α in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-α-induced apoptosis signaling. In UA cells, TNF-α-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1β converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria. J. Cell. Physiol. 174:179–185, 1998. © 1998 Wiley-Liss, Inc.     

Endothelial cells undergo morphological, biomechanical, and dynamic changes in response to tumor necrosis factor-α

The immune response triggers a complicated sequence of events, one of which is release of the cytokine tumor necrosis factor-α (TNF-α) from stromal cells, for example monocytes and macrophages. In this work we investigated the biophysical effects of TNF-α on endothelial cells (ECs), including changes in cell morphology, biomechanics, migration, and cytoskeletal dynamics. We found that TNF-α induces a wide distribution of cell area and aspect ratio, with these properties increasing on average during treatment. Interestingly, aspect ratio peaks after approximately 10?h of exposure to TNF-α, corresponding also to a peak in exerted traction forces. Meanwhile, ECs treated with TNF-α soften, and we associate this with significant increases in estimated cellular volume. In addition, our evaluation of migratory dynamics revealed an inverse correlation between cell aspect ratio and migration speed after TNF-α treatment, suggesting that cell shape may be an important functional regulator of EC migration during an inflammatory response. Finally, we addressed the basic mechanics of how the reorganization of F-actin filaments occurs during TNF-α treatment, and observed a dynamic shift of existing actin filaments. Together, our results suggest a functional link between EC morphology, biomechanics, migration, and cytoskeletal dynamics during an inflammatory response.     

Triglyceride enhances susceptibility to TNF-α-induced cell death in THP-1 cells

Monocytes and macrophages play a major role in atherosclerosis development. Previously, we found that triglyceride (TG) promoted cell death of PMA-differentiated THP-1 macrophages. In this study, we compared the responsiveness of THP-1 monocytes and PMA-differentiated THP-1 macrophages to TNF-α-induced cell death. We found that, whereas THP-1 monocytes were TNF-α-resistant, THP-1 macrophages were sensitive to TNF-α-induced cell death. THP-1 monocytes treated with TG underwent cell death beginning at 24 h and addition of TNF-α further increased cell death. Based on these observations, we hypothesized that TG-induced differentiation of THP-1 monocytes into THP-1 macrophages, subsequently allowing sensitivity to TNF-α. To determine if TG could induce differentiation of THP-1 monocytes into THP-1 macrophages, we examined the mRNA expression levels of the macrophage-specific markers, CD11b, CD18, CD36 and CD68, by RT-PCR analysis. Our results show that expression of CD11b, CD36 and CD68 increased in TG-treated THP-1 monocytes in a dose- and time-dependent manner; furthermore, TNF-α expression was upregulated in TG-treated THP-1 monocytes. We have concluded that TG induces differentiation of THP-1 monocytes into macrophages concomitant with the production of TNF-α and increased sensitivity to TNF-α-dependent cell death.     

慢性肾小球肾炎患者治疗前后血清CRP和VEGF浓度变化及临床意义

目的：研究慢性肾小球肾炎（CNG）中血清C反应蛋白（CRP）和血管内皮生长因子（VEGF）的浓度变化及其临床意义。方法：采用ELISA法测定35例正常对照组与41例慢性肾小球肾炎患者治疗前后血清IL-6和VEGF的浓度,同时放射免疫分析法测定血清TNF-α浓度,免疫比浊法测定血清CRP与尿Alb浓度。结果：①治疗前后CNG患者血清中IL-6、TNF-α和CRP较正常对照组均显著升高（P〈0.05或P〈0.01）,但治疗后IL-6、TNF-α和CRP水平显著低于治疗前（P〈0.01）,且血清CRP与IL-6和TNF-α呈正相关（P〈0.01）。②治疗后,CNG患者血清VEGF水平与尿Alb含量较治疗前明显降低（P〈0.05或P〈0.01）,仍显著高于正常对照组（P〈0.05或P〈0.01）,且血清VEGF与尿Alb水平呈正相关（P〈0.01）。结论：CRP、IL-6和TNF-α参与了CNG患者慢性炎症反应,VEGF则与蛋白尿的产生密切相关,治疗前后血清CRP和VEGF检测对于慢性肾小球肾炎的病情了解及临床疗效评估均具有重要的临床价值。     

Secretion of TNF-α from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M-CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF-α) assay. The present study has been designed to investigate whether the production of TNF-α, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF-α showed that the polyanion induced TNF-α production by macrophages. The secretion of TNF-α from the cells reached a maximum at 3-6 h, and then showed a slight decline. Northern blotting of TNF-α mRNA showed that the amount of TNF-α reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF-α mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-α mRNA, which is then followed by translation of TNF-α.     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

急性高原肺水肿患者血清中VEGF,TNF-α,IL-6及NO的含量变化

目的：检测高原肺水肿患者发生及转归过程中内皮生长因子（VEGF）,肿瘤坏死因子-α（TNF-α）,白介素-6（IL-6）和一氧化氮（NO）的含量变化,探讨高原肺水肿发生发展可能的病理生理机制。方法：收集10例高原肺水肿患者治疗前和转归后的血清样本,采用酶联免疫法和硝酸还原测定VEGF,TNF-α,IL-6和NO的含量。结果：VEGF在高原肺水肿发病时血清的含量为（167.9±26.5）pg/ml,而转归之后其含量显著降低为（53.1±17.0）pg/ml,（P〈0.01）;TNF-α在血清中的含量从发病时的（86.2±24.1）pg/ml减少到转归后的（29.2±6.8）pg/ml,（P〈0.05）,有显著性差异。而IL-6的水平也从发病时的（32.3±16.5）pg/ml变化为转归后的（12.5±8.0）pg/ml,虽然有下降的趋势,但没有统计学差异。而高原肺水肿患者血清中的NO水平从发病时的（33.8±3.3）μmol/L明显的升高到转归后的（74.1±6.2）μmol/L,（P〈0.01）。结论：VEGF,TNF-α,IL-6和NO参与了高原肺水肿的发生和转归过程。     

The adipose triglyceride lipase,adiponectin and visfatin are downregulated by tumor necrosis factor-α (TNF-α) in vivo

Inflammatory cytokines have been linked to obesity-related insulin resistance. To investigate the effect of TNF-α, an inflammatory cytokine, on insulin action, C57BL/6J mice were treated with TNF-α for 7 days after which we examined the in vivo effects of TNF-α on glucose tolerance and insulin sensitivity with IV glucose tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, we analyzed the in vivo effect of TNF-α on several metabolism-related genes and adipocytokines implicated in the development of insulin resistance. TNF-α treatment resulted in markedly increased fasting blood glucose, insulin and free fatty acids (FFA) levels and reduced glucose tolerance. During the clamps, the rates insulin-stimulated whole body (G_Rd) and skeletal muscle glucose uptake (MGU) and insulin’s ability to suppress hepatic glucose production (HGP) were decreased in TNF-α treated animals, indicating insulin resistance. In addition, both PPARγ and ATGL mRNA expression in adipose tissues as well as ATGL protein levels in plasma were downregulated. Moreover, adipose mRNA expression and plasma protein levels of adiponectin and visfatin were significantly down-regulated. We conclude that the alterations of PPARγ, ATGL, adiponectin and visfatin may contribute to the development of insulin resistance mediated by TNF-α.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

TNF-α刺激大鼠骨髓间充质干细胞的机制研究

目的：研究肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）刺激大鼠骨髓间充质干细胞（marrow-derived mesenchymalstem cells,MSCs）的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞（MSCs）,通过酶联免疫吸附剂测定法（enzyme linked immunosorbent assay,ELISA）观察比较不同组别细胞的生长因子分泌和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活（P〈0.05）;同时TNF-α刺激组与TNF-α＋NF-κB抑制剂组比较,TNF-α＋NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制（P〈0.05）。结论：NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。     

Interleukin-1 superfamily member,interleukin-33, in periodontal diseases

Interleukin (IL) -33 is a nuclear protein that is released from damaged cells and acts as an alarmin. We investigated the expression of IL-33 in human gingival fibroblasts after stimulation by tumor necrosis factor alpha (TNF-α). Human periodontal tissue samples were collected and fixed in phosphate-buffered 4% formalin in saline and processed to paraffin blocks. TNF-α was immunostained in samples of ten periodontitis patients and ten controls. Human gingival fibroblasts were isolated using an explant culture technique. The influence of TNF-α on IL-33 in gingival fibroblasts was analyzed using enzyme-linked immunosorbent assay (ELISA). The number of TNF-α positive cells was significantly greater in periodontitis samples than in controls. TNF-α was located mainly in macrophage- and fibroblast-like cells, vascular endothelial cells and epithelial cells. Analysis of IL-33 expression in cell culture lysates showed that TNF-α induced IL-33 in cultured gingival fibroblasts. Periodontitis samples are characterized by Th2 cell dominance, which has been linked to anti-inflammatory responses and periodontal repair. TNF-α-induced IL-33 may link inflammation directly to the IL-33-dependent stimulation of Th2 cytokine producing cells and participate in the induction of lymphocytes, which results in protective, anti-inflammatory and reparative responses.