Effect of lymphotoxin and tumor necrosis factor on endothelial and connective tissue cell growth and function

In an approach to understand the immune basis of human vascular and fibrotic disorders, the effects of recombinant human tumor necrosis factor alpha (rTNF) and lymphotoxin (rLT) on the in vitro growth and function of vascular and connective tissue cells were studied. Both rTNF and rLT stimulated fibroblast growth and protein, fibronectin, and collagen synthesis in dose-dependent fashion. In contrast, endothelial cell (EC) growth was inhibited by both cytokines; true EC cytotoxicity was seen at high concentrations (greater than or equal to 500 mu/ml). Addition of recombinant interferon-gamma markedly enhanced EC cytotoxicity while the growth factor beta-transforming growth factor reversed EC growth inhibition. Both rTNF and rLT stimulated factor VIII-Ag synthesis by EC. These contrasting effects of rTNF and rLT on fibroblast and endothelial cell growth and function in vitro are intriguing because they are the same contrasting effects observed in vivo in connective tissue and vascular disorders, raising the possibility of a role for these cytokines in these disorders. Study of the in vitro and in vivo mechanisms of these diverse effects may contribute to the understanding of certain human disorders characterized by endothelial injury and fibroblast activation leading to fibrosis.     

Interaction of tumor necrosis factor and lymphotoxin with model lipid membranes

TNF and LT are two pleiotropic lymphokines produced mainly by activated macrophages and lymphocytes. TNF has recently been shown to damage liposome membranes. Here, we have tested recombinant TNF and LT (rTNF and rLT) on high-resistance planar lipid bilayers and assessed changes in transmembrane current. Under the conditions tested (pH range 4.5-8), membrane permeability was not altered by these cytokines alone or in combination with gamma-interferon, in marked contrast to previously published results. Moreover, less than 5% of rTNF and rLT bound to liposomes even under low pH conditions. These results suggest TNF and LT must interact with other cellular factors (perhaps receptors) in order to mediate their activity.     

Endothelial cell activation induced by tumor necrosis factor and lymphotoxin.

Alterations in the morphology and histochemistry of vascular endothelial cells (EC) have been repeatedly observed at sites of chronic inflammation and immune reactions. These changes, which are most prominent in the EC postcapillary venules present in areas with large lymphocytic infiltrates, include the acquisition of a columnar or cuboidal morphology, the development of ribonuclease-sensitive metachromasia, and an increase in intracellular organelles. Thus, EC at sites of inflammation appear to be activated and to demonstrate increased metabolic activity. This study reports that both tumor necrosis factor-alpha (TNF) and lymphotoxin (LT) can activate cultured human umbilical vein EC, as measured by: 1) increased adhesiveness for lymphocytes, 2) increased cell metabolism, as measured by RNA and protein synthesis, and 3) increased cell volume. Although gamma interferon (IFN-gamma) and interleukin-1 (IL-1) have been shown previously to stimulate EC adhesiveness for lymphocytes, these two cytokines had only marginal effects on EC RNA and protein synthesis, and both caused a decrease in EC volume. These findings suggest that TNF and LT play a role in the type of activation of EC in vivo that leads to the development of tall endothelium and increased lymphocyte emigration.     

In vivo immunohistochemical identification of tumor necrosis factor/cachectin in human lymphoid tissue.

To find an alternative approach to the in vivo detection of tumor necrosis factor/cachectin (TNF alpha), an immunohistochemical method to identify TNF alpha in histologic sections was developed. This method employs the streptavidin-biotin immunoperoxidase technique, and TNF alpha-specific monoclonal and polyclonal antibodies, on cryostat sections of fresh frozen human lymphoid tissue. Staining was evident in most specimens displaying follicular hyperplasia, but was absent from histologically normal tissue. Both tingible body macrophages and follicular dendritic reticulum cells appeared from phenotype analysis in serial sections and by double staining experiments to constitute the main source of TNF alpha. This technique complements other systemically oriented assays that may fail to detect significant in vivo TNF alpha production and activity at a cellular level.     

Tumour necrosis factor receptor distribution in human lymphoid tissue.

The nature and location of cells responding to tumour necrosis factor-alpha (TNF-alpha) were investigated in situ by immunohistochemistry using monoclonal antibodies (mAb) directed against the p75 and p55 proteins of the TNF receptor. Receptor expression was found in the thymus and secondary lymphoid tissues. In the thymus the p75 receptor was confined to medullary lymphoblasts and dendritic cells, which co-stain with the Tac protein of the interleukin-2 (IL-2) receptor. In lymph nodes and other secondary lymphoid tissues, the p75 receptor was expressed on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal centre dendritic reticulum cells (DRC), which is the main site of TNF-alpha production. TNF receptor (TNFR) proteins were up-regulated in reactive hyperplasia together with increased TNF-alpha expression. Surprisingly, no TNFR was detectable on non-lymphoid tissues. The species specificity of these TNFR antibodies was high: whereas the antibodies cross-reacted with epitopes in non-human primates, no immunoreactivity was detected in lower animal species, e.g. dog, rabbit and rodents. The data presented suggest that TNF-alpha, which is produced by germinal centre DRC, might regulate an in vivo immune response through autocrine and paracrine pathways, e.g. through the p55 and p75 receptor proteins, which are expressed at different sites of the lymphoid tissue.     

癌组织衍生肿瘤坏死因子抑制因子的初步研究

将３０份手术切除的新鲜结直肠癌组织块直接作原代培养４８ｈ后，观察培养上清中免疫抑制因子的存在及特性。结果表明：此癌组织培养上清可抑制人外周血单个核细胞（ＰＢＭＣ）产生肿瘤坏死因子（ＴＮＦα）并抑制重组人肿瘤坏死因子（ｒｈＴＮＦα）杀伤靶细胞的活性，两种作用均呈剂量依赖性。对抑制ｒｈＴＨＦα杀瘤活性的因子初步纯化的结果证实，其为分子量约４０ＫＤ的蛋白质。此外，为保证结直肠癌组织培养不污染，本工作对癌     

Distinct roles for lymphotoxin-α and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue

Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LTα^?/? and TNF^?/? mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen. PP development in TNF^?/? mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.     

Tumor necrosis factor receptors in lymphoid tissues and lymphomas. Source and site of action of tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF alpha), which is produced by germinal center dendritic reticulum cells (DRC) in lymphoid tissue, plays a regulatory role in a local immune response. However no information is available on the nature and location of cells responding to this cytokine. Thus TNF receptor distribution was investigated in situ by immunohistochemistry using monoclonal antibodies directed against the p75 and p55 receptor proteins. Receptor expression was unique and restricted to the lymphoreticular tissue. The p75 receptor was found on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal center DRCs, which are the main site of TNF alpha production. The two receptor proteins were expressed on distinct cell populations of the lymphoid system and no coexpression was observed. Preliminary results indicate that TNF receptor (TNFR) expression is regulated; Upregulation of TNFR proteins was found in reactive hyperplasia together with increased TNF alpha expression. In lymphoproliferative disorders, expression of the p75 receptor and TNF alpha was found mainly in high-grade malignant non-Hodgkin lymphomas. In summary, TNF alpha produced by germinal center DRCs might regulate an in vivo immune response through autocrine and paracrine pathways. Thus TNF alpha might signal, through the distinct TNFR proteins, the p55 and p75 receptor, which are expressed on different cell types in lymphoid tissue.     

Production of lymphotoxin and tumour necrosis factor by a T-cell hybridoma

It is known that two major cytotoxins, lymphotoxin (LT) and tumour necrosis factor (TNF), are produced by T cells and macrophages, respectively. We report in this paper that a human T-cell hybridoma, AC5-8, produces TNF in addition to LT depending on the kind of stimuli used. When phorbol myristate acetate (PMA) and concanavalin A (Con A) or PMA alone were used to induce AC5-8 cells to secrete a cytotoxin, we found that the cytotoxin secreted was antigenically indistinguishable from LT. On the other hand, when AC5-8 cells were activated by PMA and A23187, they secreted another cytotoxin which was antigenically indistinguishable from TNF, in addition to LT. These two cytotoxins could be separated by affinity chromatography on a column of Con A-Sepharose. A cytotoxin, which did not bind to the Con A-Sepharose column and showed about 70-95% of the total cytotoxic activity secreted from AC5-8 cells stimulated with PMA and A23187, was found to be antigenically identical to TNF. The other cytotoxin, which bound to the Con A-Sepharose column and showed only 5-30% of the total activity induced by PMA and A23187, was found to be antigenically identical to LT.     

Recombinant human alpha lymphotoxin (tumor necrosis factor-beta) induces peripheral neutrophilia and lymphopenia in the rat.

Recombinant human alpha lymphotoxin (rLT) administered intravenously to Lewis rats induces peripheral neutrophilia and lymphopenia in a dose-response dependent fashion. A dose of 30,000 units of rLT induced a neutrophilia (1589 +/- 326 to 5554 +/- 1050 neutrophils/cu mm) and lymphopenia (10,368 +/- 992 to 4636 +/- 878 lymphocytes/cu mm) at 2 hours after injection that was highly significant (P less than 0.001 and P less than 0.001, respectively) in comparison with vehicle controls. The kinetics of the neutrophilia that peaked at 2 hours as well as of the lymphopenia were highly reminiscent of the neutrophilia and lymphopenia following intravenous administration of either recombinant human interleukin-1 (IL-1) alpha or beta to rats. The peripheral neutrophilia was accompanied by a significant depletion of bone marrow neutrophils (P less than 0.001), as is also known to occur after administration of IL-1. Systemic blood pressure was not affected by rLT, which suggested that the changes in circulating leukocyte subsets were not attributable to hemodynamic changes nor to the hemodynamic-change-related release of adrenal hormones. Adrenalectomy did not alter the rLT-induced neutrophilia or lymphopenia, which suggested that rLT does not mediate its hematologic effects on peripheral blood leukocytes via the release of adrenal hormones. Pretreatment of rats with dexamethasone, indomethacin, or aspirin also did not alter rLT-induced neutrophilia or lymphopenia, which suggested that rLT-induced hematologic effects were not mediated via arachidonic acid metabolites, in stark contrast to IL-1 induced neutrophilia, which is inhibited by both dexamethasone and indomethacin.     

肿瘤坏死因子对体外培养的血管内皮细胞的作用

在体外研究了肿瘤坏死因子对人脐静脉血管内皮细胞的作用。在体外培养的ＨＶＥＣ中加入人重组的ＴＮＦａ，终浓度为４００Ｕ／ｍｌ，培养７２小时后取贴壁细胞及培养液进行研究。结果发现，与对照组相比，ＴＮＦ组细胞有如下变化：１、光镜观察可见ＮＴＦ组细胞明显拉长，电镜观察发现细胞表面微绒毛减少，胞质中次级溶酶体增多，线粒体肿胀，变性，髓样小体出现。２．ＴＮＦ对ＨＶＥＣ的增殖有抑制作用。３．细胞表面纤维连接蛋白的     

肿瘤坏死因子-α对肝星状细胞增殖与凋亡的影响

目的:探讨肿瘤坏死因子-α(TNF-α)在肝纤维化发病中的作用。方法:采用TNF-α对体外培养的HSC进行干预, 通过流式细胞术、电镜及TUNEL等方法, 对HSC的增殖与凋亡进行研究。结果:①流式细胞术显示:TNF-α各组的G₀/G₁期细胞比率明显高于对照组, 而S期细胞比率明显低于对照组, 各组的细胞增殖指数均明显低于对照组;在凋亡方面, TNF-α各组的HSC凋亡率明显高于对照组, TNF-α各组HSC中抗凋亡基因bcl-2的表达明显低于对照组, 而促凋亡基因bax的表达明显高于对照组。②TUNEL分析显示:TNF-α(2.0μg/L)组的HSC凋亡率为18.7%±2.5%, 而对照组为5.3%±1.2%, 两者之间有显著差异。结论:TNF-α能够阻止HSC从G₀/G₁期进入S期, 从而具有抑制HSC增殖的作用;TNF-α能够降低HSC中bcl-2的表达、提高bax的表达, 这可能与其促进HSC凋亡有关。     

银屑病外周血T细胞亚群与肿瘤坏死因子的研究

本文以结晶紫染料摄入法,检测52例寻常型银屑病患者内毒素诱导的外周血单个核细胞(PBMCs)产生肿瘤坏死因子(TNF)活性,并用T 细胞表型(T_1、T_4、T_8)单克隆抗体间接免疫荧光法,检测患者外周血T 细胞及其亚群的百分率。结果寻常型银屑病患者外周血TNF 诱生活性(6.13±2.41U/ml)、T_4细胞百分率(46.31±6.31%)及T_4/T_(?)比值(1.54±0.27)均显著低于正常人对照组(P<0.01),而T_(?)细胞百分率(30.33±2.12%)则明显增高(P<0.01)。表明银屑病患者存在细胞免疫功能缺陷;TNF 产生能力低下和T_4/T(?)的比例失调与银屑病的发病密切相关。     

Independent protective effects for tumor necrosis factor and lymphotoxin alpha in the host response to Listeria monocytogenes infection

Although the essential role of tumor necrosis factor (TNF) in resistance to Listeria monocytogenes infection is well established, the roles of the related cytokines lymphotoxin alpha (LTalpha) and lymphotoxin beta (LTbeta) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTalpha, and LTbeta in the host response to Listeria. To overcome the lack of peripheral lymph nodes in LTalpha(-/-) and LTbeta(-/-) mice, bone marrow chimeras were constructed. TNF(-/-) and LTalpha(-/-) chimeras that lacked both secreted LTalpha(3) and membrane-bound LTalpha(1)beta(2) and LTalpha(2)beta(1) were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTbeta(-/-) chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. The Listeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTalpha(-/-) and LTbeta(-/-) chimeras, WT chimeras, and TNF(-/-) and WT mice). TNF(-/-) mice and LTalpha(-/-) chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF(-/-) and LTalpha(-/-) chimeras. Therefore, in addition to TNF, soluble LTalpha(3) plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.     

Highly sensitive enzyme immunoassays for antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Two 'inverse sandwich' enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), respectively. In these one-step assays, antibodies present in the sample linked antigen which had been covalently coupled to horseradish peroxidase to antigen bound to a solid phase (microtiter plates). The limits of detection of the assays were lower than those of neutralization bioassays; antibodies to TNF-alpha and TNF-beta being detected at concentrations as low as 2 ng/ml and 0.5 ng/ml, respectively, and no cross-reactivity was observed. The advantages of these ELISAs over other assay methods currently in use for the detection of antibodies include: (i) the convenience of the microtiter plate format and its suitability for testing large numbers of samples; (ii) the absence of radioactive tracers and precipitation steps; (iii) the high stability of the reagents; (iv) the avoidance of second antibodies and, thus, the possibility of testing samples from various species without modification of the assay and (v) the ability to detect low-affinity antibodies due to the absence of competitive reactions. The assays may be used without modification for the detection of antibodies in serum samples from both man and laboratory animals as well as in other samples such as hybridoma supernatants.     

Chemotactic activity of lymphotoxin and tumour necrosis factor alpha for human neutrophils.

Lymphotoxin (TNF beta) and tumour necrosis factor alpha (TNF alpha) stimulate locomotion and chemotaxis of human blood neutrophils as measured in three assays. Both cytokines stimulate morphological polarization of neutrophils in suspension; both stimulate locomotion of neutrophils into micropore filters; both cause orientation of neutrophils towards a gradient source. Orientation in a gradient suggests a chemotactic effect. The action of both cytokines is similar but is not as strong as that of formyl peptide used as a positive control. Myelomonocytic cell lines (U937 and HL-60) develop responsiveness to formyl peptides on maturation but not to TNF alpha or beta.     

Effects of in vivo 'priming' on endotoxin-induced hypotension and tissue injury. The role of PAF and tumor necrosis factor.

Exogenously administered tumor necrosis factor-alpha (TNF) and bacterial endotoxin (LPS) induce shock and tissue injury. Here, the authors studied the effect of endogenous TNF on LPS-induced hypotension and tissue injury and investigated the role of PAF in these responses. Rats were primed with intraperitoneal injection of zymosan 24 hours before, or Bacillus Calmette-Guérin (BCG) 12 to 15 days before intravenous injection of low dose (0.5 mg/kg) LPS. It was found that nonprimed animals showed mild hypotension and moderate leukopenia in response to LPS. In contrast, zymosanprimed rats developed shock and marked leukopenia, and more severe bowel injury than nonprimed rats. The authors then showed that, following LPS injection, zymosan-primed animals had higher TNF and platelet-activating factor (PAF) levels than nonprimed rats. Pretreatment of the animal with PAF antagonist, SRI 63-441, markedly ameliorated the hypotension and tissue injury. Interestingly, BCG-primed rats did not show aggravation of LPS-induced hypotension. Only TNF (but not PAF) level in these animals was increased. Thus, it appears that TNF release alone, without a sufficient increase in PAF, is incapable of causing severe hypotension. However, most of the BCG-primed animals showed tissue injury, which could be prevented by pretreatment with PAF antagonist. The authors discuss the possible mechanisms of this discrepancy between systemic and local responses in BCG-primed animals.     

Effects of quinolones on tumor necrosis factor production by human monocytes.

Previous studies have shown that in lipopolysaccharide (LPS)-stimulated human monocytes, interleukin 1 (IL-1) production is altered by quinoline derivative antibiotics (quinolones), in a way which depends both on the dose and on the agents used. Given that IL-1 and tumor necrosis factor alpha (TNF) are produced in response to LPS and have some overlapping and synergistic activities, we sought to determine if TNF production was altered under the above-mentioned conditions. We investigated the effects of three quinolones: ciprofloxacin (Cip), pefloxacin (Pef) and ofloxacin (Ofl). These quinolones were found to decrease extracellular TNF production in a dose-dependent manner at concentrations higher than 25 micrograms/ml as previously described by our laboratory with regard to IL-1 production. Moreover, the order of the extracellular decrease in TNF and IL-1 induced by each drug was similar. However, in contrast to IL-1 activity, the quinolones studied also reduced cell-associated TNF. The kinetics of TNF production suggested that the quinolones affected TNF production at a very early step, probably during TNF synthesis rather than during its secretion into the extracellular medium. Furthermore, the quinolone-induced accumulation of intracellular cAMP could explain the extracellular decrease in both IL-1 and TNF production.     

Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature

Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/-), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/- mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF-induced tumor necrosis.