Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells

Abstract

Background: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. Objective: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. Materials and methods: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. Results: CD19⁺ B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3⁺ T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14⁺ monocytes. Conclusion: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.     

The potential biological and clinical significance of the soluble tumor necrosis factor receptors

The role of TNF receptors (TNF-Rs) is not limited to signal transduction but includes extracellular regulatory functions affecting systemic TNF bioavailability. This review summarizes the regulation of TNF-R shedding and its kinetics, the complex interaction between the soluble receptors and their ligand in vitro and in vivo, and the potential diagnostic, prognostic and therapeutic value of the soluble receptors in malignant, inflammatory, infectious and autoimmune disorders.     

Differential activation of phospholipases during necrosis or apoptosis: A comparative study using tumor necrosis factor and anti-Fas antibodies

Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A₂ (cPLA₂) are differentially activated during TNF-induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti-Fas antibodies as cell death stimuli showed that PLD and cPLA₂ are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers. J. Cell. Biochem. 71:392–399, 1998. © 1998 Wiley-Liss, Inc.     

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.     

NT FRZ 基因多态性与SEL 的相关性研究

本研究通过调查中国南方SLE人群和健康对照人群中TNFR2基因两个位点（nt587,nt694）的多态性频率,探讨TNFR2基因多态性是否与中国汉族SLE人群的易感性相关。结果发现128例SLE中,nt587G的等位基因频率为54个（21．1％）;而135例健康人群中nt587G的频率为35个（13．0％）;SLE组明显高于健康对照人群（P〈0．05）,携带nt587G的个体SLE发病危险性大。同时128例SLE中,舶94A的等位基因频率为41个（16．0％）;而健康人群中舶94A的频率为32个（11．9％）;两组比较无显著差异（P=0．149）。提示TNFR2基因nt587的多态性与中国南方SLE人群相关,可能通过影响TNFR2的表达而参与SLE的发病,而nt694（G—A）的多态性与中国南方SLE人群不相关。     

Cardiac lipoprotein lipase: effects of lipopolysaccharide and tumor necrosis factor

Summary Lipopolysaccharide (LPS), the active principle of certain endotoxins, protein-free perfused in rat hearts leads in 3 h to a considerable loss of lipoprotein lipase (LPL) activity. In the presence of albumin LPS has virtually no effect. Tumor necrosis factor (TNF) added instead of LPS had no effects on LPL activity during 3 hin vitro perfusion. LPS injected into rats intravenously leads within 3 h to severe toxic phenomena amongst which increased capillary permeability. This was visualized as increased rate of interstitial fluid formation in Langendorff hearts mounted 3 h after rats had been treated with LPS. LPL activity did not decline in 3 h lasting endotoxemia. Six hours after LPS injection, however, cardiac LPL activity was considerably lowered, although immunoblotting and immunohistochemistry still showed LPL protein to be present. These date indicate the presence of a considerable pool of inactive LPL protein in addition to active LPL, that can be released in the presence of heparin. The LPL activity is lowered by LPS injection after a lag phase of at least 3 h, while capillary endothelial cells are influenced more rapidly. The relatively late expression of TNF toxicity in cardiomyocytes of the intact heart is discussed.     

Tumor necrosis factor receptor superfamily members 1a and 1b contribute to exacerbation of atherosclerosis by Chlamydia pneumoniae in mice

The host immune responses that mediate Chlamydia-induced chronic disease sequelae are incompletely understood. The role of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2), in Chlamydia pneumoniae (CPN)-induced atherosclerosis was studied using the high-fat diet-fed male C57BL/6J mouse model. Following intranasal CPN infection, TNF-α knockout (KO), TNFR1 KO, TNFR2 KO, and TNFR 1/2 double-knockout, displayed comparable serum anti-chlamydial antibody response, splenic antigen-specific cytokine response, and serum cholesterol profiles compared to wild type (WT) animals. However, atherosclerotic pathology in each CPN-infected KO mouse group was reduced significantly compared to WT mice, suggesting that both TNFR1 and TNFR2 promote CPN-induced atherosclerosis.     

Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测

本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。     

Astragaloside suppresses tumor necrosis factor receptor-associated factor 5 signaling pathway and alleviates neurodegenerative changes in retinal pigment epithelial cells induced by isoflurane

Epidemiological studies showed that isoflurane, a general anesthetic widely used in surgery including those for the children, is associated with impairment of neurodevelopment and neurodegenerative diseases, such as Alzheimer’s disease (AD) and age-related macular degeneration (AMD), which are related to the accumulation of reactive oxygen species (ROS). Astragaloside (AS) is an antioxidant derivative from a traditional Chinese herbal medicine Astragalus membraneaceus Bunge. In this study, we used retinal pigment epithelial cells, which share plenty of features with neurodegenerative diseases such as AD and AMD to investigate the effect of AS. Cell cycle re-entry and proapoptosis were seen in retinal pigment epithelium (RPE) cells treated with isoflurane, which was alleviated by pretreatment of AS. Further, tumor necrosis factor receptor-associated factor 5 (TRAF5) and downstream nuclear factor-κB (NF-κB) were investigated to elucidate the molecular mechanism underlying protective effect of AS. RPE cells exposed to isoflurane expressed higher TRAF5 and NF-κB than those pretreated with AS, suggesting a critical role of TRAF5 therein. In Morris water maze (MWM) assay, Sprague-Dawley rats pretreated with AS and then exposed to isoflurane spent less time in swimming to the platform, and their TRAF5 expression was significantly lower than those received anesthesia alone. Further studies on the consequence of forced downregulation or upregulation are warranted that may employ cutting-edge technologies such as optogenetics to overcome the difficulties in manipulating expression of TRAF5. Although the link between TRAF5 and neurodegeneration requires more in-depth investigations, our study provide a novel hint on the pathological mechanism of isoflurane and suggest a potential target for eliminating persistent side effect of anesthesia.     

Protection against pulmonary oxygen toxicity by interleukin-1 and tumor necrosis factor: Role of antioxidant enzymes and effect of cyclooxygenase inhibitors

Rats injected with interleukin-1 (10 g) and tumor necrosis factor (10 g) and then exposed continuously to hyperoxia (> 99% O₂, 1 atm) survived longer, had increased lung reduced/oxidized glutathione ratios, smaller pleural effusions, less pulmonary hypertension and improv+++ed arterial blood gases. The percentage of animals surviving for 72 hours in hyperoxia increased from 8% to 94%. Although relatively small increases in glutathione redox cycle enzymes occurred four and sixteen hours following cytokine injection, dramatic increases in all major antioxidant enzymes including superoxide dismutase, glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and catalase had occurred following 72 hours of exposure to hyperoxia. The protective effect of IL-1 + TNF against lethal pulmonary O₂ toxicity could be partially inhibited by pre-injection of lysine acetylsalicylate or, less effectively, of ibuprofen.Recent studies have suggested that both IL-1 and TNF can induce manganese (mitochondrial) superoxide dismutase mRNA and protein synthesis in a variety of cell types. Preliminary studies suggest that IL-1 alone, in ample dosage, can provide protection against lethal pulmonary O₂ toxicity. Future studies should be directed toward identification of acute phase changes in lung antioxidant enzymes, surfactant proteins and/or lipid components, enzymes needed for synthesis of surfactant phospholipids, and/or other protective proteins. Additional work also needs to be done in identifying the lung cell types in which early enzyme induction occurs. These studies should provide a better understanding of mechanisms whereby protection against pulmonary O₂ toxicity can occur. An understanding of the molecular mechanisms inducing protective proteins should lead to more precise pharmacologic control of these processes.     

Effects of nickel oxide on the production of tumor necrosis factor by alveolar macrophages of rats

To evaluate the effect of green nickel oxide (NiO) on the production of tumor necrosis factor (TNF) by alveolar macrophages, alveolar macrophages were exposed to NiO in vitro and in vivo. For the in vitro study, rats alveolar macrophages were incubated with NiO on a microplate for 24 h. TNF activity in the culture supernatant was determined by the L929 bioassay. Rats alveolar macrophages cultured with 100 and 200 μg/mL of NiO in vitro induced the production of TNF, however, it was not statistically significant compared with the control that was free from NiO exposure. For exposure in vivo, rats were divided into two groups. Five were exposed to a daily concentration of 11.7±2.0 mg/m³ of NiO for an 8-hr/d, 5 d/wk, for 4 wk, and five rats (control) were kept in a cage and not exposed to NiO. Bronchoalveolar lavage was performed and the recovered alveolar macrophages were incubated on a microplate for 24 h. TNF production by exposed alveolar macrophages was significantly higher than that of controls.     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995     

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor^+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor^−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.     

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF- secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF- detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF- is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.     

Preventive effect of several drugs againstPseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: Relationship between lethality and the arachidonic cascade

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) inPseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C₄/D₄/E₄ or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.