Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane-bound TNF in experimental cerebral malaria

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of experimental cerebral malaria (CM), but the respective role of its two types of receptors has not been established. A significant increase in the expression of TNF-receptor 2 (TNFR2, p75), but not of TNFR1 (p55), was found on brain microvessels at the time of CM in susceptible animals. Moreover, mice genetically deficient for TNFR2 (Tnfr2°) were significantly protected from experimental CM, in contrast to TNFR1-deficient (Tnfr1°) mice, which were as susceptible as wild-type mice. To identify the factors involved in the protection from CM conferred by the lack of TNFR2, we assessed in both knockout and control mice the serum concentrations of mediators that are critical for the development of CM, as well as the up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the brain microvessels. No significant difference in serum levels of TNF and interferon-γ was found between infected wild-type and Tnfr1° or Tnfr2° mice. Interestingly, the pronounced ICAM-1 up-regulation and leukocyte sequestration, typically occurring in brain microvessels of CM-susceptible animals, was detected in infected control and Tnfr1° mice – both of which developed CM – whereas no such ICAM-1 up-regulation or leukocyte sequestration was observed in Tnfr2° mice, which were protected from CM. Making use of microvascular endothelium cells (MVEC) isolated from wild-type, Tnfr1° or Tnfr2° mice, we show that soluble TNF requires the presence of both TNF receptors, whereas membrane-bound TNF only needs TNFR2 for TNF-mediated ICAM-1 up-regulation in brain MVEC. Thus, only in MVEC lacking TNFR2, neither membrane-bound nor soluble TNF cause the up-regulation of ICAM-1 in vitro. In conclusion, these results indicate that the interaction between membrane TNF and TNFR2 is crucial in the development of this neurological syndrome.     

Antibody to recombinant murine tumor necrosis factor (TNF) neutralizes guinea pig TNF.

Cotton dust has been found to cause acute pulmonary inflammation and fever in humans and in a guinea pig model of byssinosis. Following 3 h inhalation of cotton dust particles, guinea pig macrophages were found to release ex vivo a factor(s) toxic to WEHI fibrosarcoma cells. The cytotoxic factor(s) was also present in the bronchoalveolar fluid. We sought to investigate the mechanism of the inflammatory response to determine whether the factor was TNF. Antibodies to murine TNF were produced by immunizing sets of rabbits using two protocols. All animals produced anti-TNF antibodies with titers of 1/1000-1/25,000. Sera from one set of animals completely neutralized the cytotoxicity of murine TNF toward the WEHI cell line. The antisera neutralized up to 93% of the cytotoxicity of guinea pig samples but only 54% of human recombinant TNF. These results identify TNF in pulmonary tissues of guinea pigs following exposure to cotton dust. Moreover, the studies indicate that rabbit antibodies to murine TNF can be used to detect the guinea pig cytokine.     

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.     

The role of the tumor necrosis factor (TNF)--Fas L and HCV in the development of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major public health problem in Egypt due to the high prevalence of hepatitis C viral (HCV) infection. The mechanism by which HCV exerts its carcinogenic effect on the liver is not yet understood. Previous research has suggested that perturbations of the Fas-Fas L tumor necrosis system could result in uncontrolled cancerous cell growth in the liver. This study aims to assess the relationship of Fas ligand (Fas L) to HCC. A total of 28 cases (HCC) and 56 controls (28 cirrhosis and 28 chronic hepatitis) were included in the study. Sera and tissue biopsies were tested for HCV antibody and HCV-RNA. Fas ligand expression in tissue was examined immunohistochemically using a rabbit purified polyclonal antibody. Levels of soluble Fas L were determined in serum by ELISA. The HCC cases were graded as: 17.9% Grade I, 32.1% Grade II, 35.7% Grade III and 14.3% were Grade IV. Among the cases, 81% had evidence of cirrhosis. All the cases and controls were positive for HCV-RNA. Tissue and serum PCR results were identical within the same subjects. Fas ligand cytoplasmic expression was more pronounced in HCC than in cirrhosis, and in cirrhosis more than in chronic hepatitis. This expression was higher with increasing grades of malignancy and in tissues adjacent to the tumor, than in those without nearby tumor. Soluble Fas L levels were higher in cases than in controls, with similar results as that of immunohistochemical expression. These results suggest that HCV and Fas ligand play a key role in hepatocarcinogenesis, consistent with the hypothesis that HCV induces overexpression of Fas ligand in the liver cells, resulting in escape from killing by the immune system cells, with subsequent uncontrolled growth of tissue and the development of malignancy.     

Induction of circulating tumor necrosis factor (TNFα) as the mechanism for the febrile response to interleukin-2 (IL-2) in cancer patients

Fever is frequently observed in cancer patients treated with high-dose recombinant human interleukin-2 (rIL-2). The preincubation of rIL-2 with polymyxin B, an antibiotic that inhibits the biologic effects of endotoxins, did not diminish the pyrogenicity of IL-2 in New Zealand rabbits, indicating that IL-2-induced fever is not due to contaminating endotoxins. In contrast to interleukin-1 (IL-1), tumor necrosis factor (TNF), and interferon , which cause fever through their effects on arachidonic acid metabolism in the hypothalamus, IL-2 was unable to induce prostaglandin E₂ synthesis in hypothalamic cells or fibroblastsin vitro, suggesting that IL-2 is not intrinsically pyrogenic. To determine if IL-2-induced fever is mediated indirectly through the generation of pyrogenic cytokines, culture supernatants from IL-2-stimulated human peripheral blood mononuclear cells were screened for the presence of pyrogens by direct injection into rabbits and by measuring the amounts of IL-1, IL-1, and TNF by specific radioimmunoassays (RIA). All three cytokines were readily detected by RIA in these supernatants, which in turn caused fever when injected into rabbits. Furthermore, in six of six cancer patients treated with rIL-2, elevated levels of TNF were detected in the plasma by RIA 2 hr after IL-2 administration. Plasma TNF levels increased from pretreatment values of 14±7 to 765±150 pg/ml 2 hr after an IL-2 injection. These results strongly implicate IL-2-induced pyrogenic cytokines, in particular TNF, as a major cause of the fever and possibly other aspects of the acute-phase response associated with IL-2 therapy.     

Resistance to silica-induced lung fibrosis in senescent rats: role of alveolar macrophages and tumor necrosis factor-alpha (TNF)

The purpose of the present study was to investigate the effect of aging on silica-induced lung toxicity. In young animals silica induced a significant increase in bronchoalveolar lavage tumor necrosis factor-alpha (TNF), lactate dehydrogenase as well as in cell numbers, which correlate with increased collagen deposition and silicotic nodules formations. In old rats, however, no changes in bronchoalveolar lavage or lung parameters were observed following silica instillation. These in vivo results were also confirmed in vitro, where silica failed to induce TNF release in alveolar macrophages obtained from old animals. This defective response to silica could be explained with defective protein kinase C translocation, due to a reduction in its anchoring protein RACK-1 with aging. Overall, these data indicate that the understanding of the molecular mechanisms undelaying toxicity is crucial to define the influence of age on the toxic response and progression of the disease.     

Identification of single nucleotide polymorphisms in the tumor necrosis factor (TNF) and TNF receptor superfamily in the Korean population

The tumor necrosis factor (TNF) and TNF receptor (TNF-TNFR) superfamily plays crucial roles in immune regulation and host immune responses. The superfamily has been also associated with many immune-mediated diseases such as asthma, rheumatoid arthritis, inflammatory bowel disease, and diabetes. In order to investigate genetic variants of the TNF-TNFR superfamily, a total of 63 known single nucleotide polymorphisms (SNPs) in the coding region (cSNPs) of the TNF-TNFR superfamily genes were selected from the public SNP database. Among 63 cSNPs tested in this study, only 24 SNPs (38%) were validated to be polymorphic in the Korean population by primer extension-based SNP genotyping. By means of the new enhanced single strand conformational polymorphism (SSCP) method, we also identified a total of 78 SNPs, including 48 known SNPs and 30 novel SNPs, in the 44 human TNF-TNFR superfamily genes. The newly discovered SNPs in the TNF-TNFR superfamily genes revealed that the Korean population had very different patterns of allele frequency compared with African or white populations, whereas Korean allele frequencies were highly similar to those of Asian (correlation coefficient r = 0.88, p < 0.046). A higher similarity of allele frequency was observed between Korean and Japanese populations (r = 0.90, p < 0.001). The validated SNPs in the TNF-TNFR superfamily would be valuable for association studies with several immune-mediated human diseases.     

Effect of immunostimulants and antitumor agents on tumor necrosis factor (TNF) production

OK-432, a lyophilized preparation of Streptococcus pyogenes, showed a priming activity for TNF production in mice, associated with an increase of spleen weight. PSK, a protein-bound polysaccharide preparation from Coriolus versicolor, did not show such activity. Both OK-432 and PSK potentiated the TNF production in mice primed with Corynebacterium parvum (CP) and challenged with Escherichia coli endotoxin (LPS). Cytotoxic antitumor agents of 5-fluorouracil (5-FU), cyclophosphamide (CY) and bleomycin (BLM) suppressed TNF production in mice primed with CP and challenged with LPS. TNF production suppressed by 5-FU, CY and BLM was partially restored by the combined treatment with OK-432 or PSK. These results suggest that the administration of cytotoxic antitumor agents suppresses the intrinsic TNF production in cancer patients, and the combined use of immunostimulants such as OK-432 and PSK is advantageous in restoring TNF production suppressed by cytotoxic antitumor agents.     

Malaria, blood glucose, and the role of tumour necrosis factor (TNF) in mice

Hypoglycaemia in falciparum malaria is associated with a poor prognosis and is correlated with mortality. High levels of serum TNF are also correlated with disease severity and mortality, and it has been suggested that TNF may cause the hypoglycaemia. However hypoglycaemia in mice infected with Plasmodium chabaudi or the lethal strain of P. yoelii YM is related to hyperinsulinaemia. Its development was not prevented by treatments which diminished TNF activity or production without affecting levels of plasma insulin. Conversely, it was inhibited by diazoxide, which inhibited insulin secretion but did not affect TNF production. Furthermore, in mice exhibiting neurological symptoms during infection with P. berghei, blood glucose concentrations were significantly raised when TNF levels were high, and TNF levels in the spleen were highest of all in non-lethal P. yoelii infections in which hypoglycaemia does not occur. Administration of human rTNF to normal animals caused an increase rather than a drop in blood glucose levels. Mice transgenic for human TNF did not develop hypoglycaemia when infected with P. yoelii YM, but showed signs of insulin resistance. In line with current views on the role of TNF in obesity and the control of glucose homeostasis, we conclude that the hypoglycaemia of malaria is not caused by increased levels of TNF, which may in fact be beneficial, but is secondary to a hyperinsulinaemia that is probably stimulated directly by products of the parasite.     

TNFβ基因微卫星多态性与Graves病的相关性研究

目的 研究Graves病患者TNF(tumor necrosis factor)β基因第一内含子的微卫星多态性(TNFc)，分析TNFc与Graves病发病的关联，进一步探讨Graves病的发病机制。方法 选取Graves病实验组和正常对照组患者各36例，应用聚合酶链反应(PCR)技术，扩增具有微卫星多态性的TNFβ基因第一内合于，通过聚丙烯酰胺凝胶电泳，分析两组的基因频率和基因型频率。结果 TNFc微卫星多态性合有两个等位基因(TNFcl和TNFc2)及三种基因型(纯合于TNFclcl和TNFc2c2，亲合于TNFclc2)；Graves病实验组的TNFc2基因频率高于正常对照组，有显著性差异(x^2=4，02，P＜0．05)，TNFclcl基因型频率在Graves病实验组与正常对照组无显著性差异(X^2=2．72，P＞0．05)。结论 TNFc2等位基因与Graves病发病的易感性有关联，TNFclcl基因型在Graves病发病的遗传基础中不起重要作用。     

Allergic lung inflammation is mediated by soluble tumor necrosis factor (TNF) and attenuated by dominant-negative TNF biologics

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine consisting of soluble and transmembrane forms, with distinct roles in inflammation and immunity. TNF is an important factor in allergic airway inflammation. However, the disparate functions of soluble (sol) and transmembrane (tm) TNF in lung pathology are not well understood. Our aim was to assess the activities of solTNF and tmTNF in murine models of allergic airway disease, and to evaluate the efficacy of solTNF-selective inhibition. We used ovalbumin sensitization and challenge of TNF knockout, tmTNF knockin, and wild-type C57BL/6 mice to distinguish differences in airway inflammation and hyperreactivity mediated by solTNF and tmTNF. Functions of solTNF and tmTNF in hyperresponsive, wild-type Balb/c mice were assessed by comparing dominant-negative anti-TNF biologics, which antagonize solTNF yet spare tmTNF, to etanercept, a nonselective inhibitor of both TNF forms. Responses in transgenic C57BL/6 mice demonstrated that solTNF, and not tmTNF, is necessary to drive airway inflammation. In Balb/c mice, dominant-negative TNF biologics administered during immunization decreased the recruitment of eosinophils and lymphocytes into the bronchoalveolar space and lung parenchyma, reduced specific serum IgE, goblet-cell hyperplasia, and eosinophilic inflammation, and suppressed methacholine-induced airway hyperreactivity. Concentrations of IL-5, CCL5/RANTES, CCL11/eotaxin, and CCL17/TARC were also reduced in bronchoalveolar lavage. Dominant-negative TNFs reduced lung eosinophilia, even when given only during antigen challenge. The selective inhibition of soluble TNF suppresses inflammation, hyperreactivity, and remodeling in transgenic and wild-type murine models of allergic airway disease, and may offer safety advantages in therapies that preserve the immunoprotective functions of transmembrane TNF.     

Histamine (HA) suppresses the production of tumor necrosis factor alpha (TNFα) in cultured astroglial cells

Inflammation Research -     

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids

Objective and Design: Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. Materials and Treatment: As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5×10⁻⁹ M) in the presence or absence of dexamethasone (10⁻⁹–10⁻⁶ M). Methods: Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. Results: During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. Conclusions: These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action. accepted by K. Brune and M. J. Parnham     

Polymorphism within the tumor necrosis factor alpha (TNF) promoter region in patients with ankylosing spondylitis

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.     

Sorting soluble tumor necrosis factor (TNF) receptor for storage and regulated secretion in hematopoietic cells

Hematopoietic cells contain secretory lysosomes that degranulate at sites of inflammation. We envisage that secretory granules can act as vehicles for targeting inflammatory sites, including malignancies, and thereafter, locally release therapeutically active agents to these sites. Exogenous proteins, such as the soluble tumor necrosis factor receptor 1 (sTNFR1), have been shown previously to be targeted to secretory lysosomes [1]. In this work, we asked whether exogenous, secretory lysosome-targeted proteins were subject to regulated secretion. sTNFR1-transmembrane (tm)-cytosol-sorting signal (Y) and sTNFR1-tm-Y-enhanced green fluorescent protein (egfp) were expressed in rat basophilic leukemia cell clones having different secretory capacities. sTNFR1-tm-Y was targeted directly from the Golgi to secretory lysosomes, followed by generation of membrane-free sTNFR1, whose secretion could be triggered by a Ca2+ ionophore or immunoglobulin E receptor activation. In contrast, sTNFR1-tm-Y-egfp was targeted to the plasma membrane and then subjected to endocytosis and presumably, secretory lysosome targeting, as judged by results from antibody ligation and cell-surface biotinylation. Activation of protein kinase C with phorbol ester promoted ectodomain shedding at the cell surface, resulting in sTNFR1 release from sTNFR1-tm-Y-egfp. These results support a concept for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.     

Polymorphisms at tumor necrosis factor (TNF) loci are not associated with Chagas' disease

Abstract: The aim of this study was to investigate whether the polymorphisms at the TNF loci are associated with Chagas' disease. We determined the TNFA (positions -308, -244 and -238) and TNFB genotypes in a sample of 85 serologically positive chagasic individuals and in 87 healthy controls from a Peruvian population where Trypanosoma cruzi infection is endemic. Patients were subdivided according to the presence ( n =33) or absence ( n =52) of chagasic chronic cardiomyopathy, in order to search for genetic differences associated with this pathological condition. No significant differences either between patients and controls or between asymptomatic and cardiomyopath-ic individuals were observed with respect to TNFA or TNFB polymorphisms when these were considered individually or as extended haplotypes.     

Tumor necrosis factor (TNF) is induced in mice by Candida albicans: role of TNF in fibrinogen increase.

One intraperitoneal dose of Candida albicans (10(8) CFU) caused a chronic (longer than 2 months), significant elevation of plasma fibrinogen levels (Clauss method) in mice of strain C3H/HeN. Even a small dose (10(6) CFU) resulted in a significant increase in fibrinogen level for 5 days following injection, whereas other blood parameters (leukocytes, erythrocytes, platelets, hematocrit, hemoglobin, blood urea nitrogen, aspartate aminotransferase, albumin, alkaline phosphatase, antithrombin III, glucose, calcium, and total protein) measured by standard methods were normal. Blood taken during this period was negative for C. albicans. The role of tumor necrosis factor (TNF) in C. albicans infections was investigated by measuring the fibrinogen response after the administration of C. albicans or recombinant mouse TNF-alpha. Both challenges resulted in an elevated fibrinogen level. When polyclonal antibodies to mouse TNF-alpha were given prior to challenge with C. albicans or mouse TNF-alpha, the fibrinogen increase was significantly inhibited. C. albicans injections were found to significantly elevate endogenous TNF levels in mice (enzyme-linked immunosorbent assay). It was concluded that C. albicans induces TNF in the mouse. Furthermore, these data give evidence which supports a relationship between TNF and the fibrinogen increase induced by C. albicans.     

Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent that specifically induces apoptosis in broad-spectrum tumor cell lines, meanwhile leaving normal cells unaffected. Unfortunately, the clinical development of TRAIL was hampered, and could be attributed to its instability, bioavailability or poor delivery. Although N-terminal specific PEGylation provides a means to improve the pharmacokinetic and stability of TRAIL, it took a bit longer time to accomplish the PEGylation process than expected. We therefore designed another PEGylation approach, mutated Cys-SH site-specific PEGylation, to conjugate methoxypoly(ethylene glycol) maleimide (mPEG-MAL) with TRAIL (95–281) mutant N109C. Asn-109 was chosen as the PEGylated site for it is a potential N-linked glycosylation site. It was shown that ∼90% TRAIL mutant N109C could be PEGylated by mPEG-MAL within 40 min. And mPEG_MAL-N109C was revealed to possess superior in vitro stability and antitumor activity than N-terminal specifically PEGylated TRAIL (114–281) (mPEG_ALD-TRAIL_114–281). What's more, mPEG_MAL-N109C exhibited more therapeutic potentials than mPEG_ALD-TRAIL_114–281 in tumor xenograft model, benefitting from better drug delivery and bioavailability. These results have demonstrated mutated Cys-SH specific PEGylation is an alternative to site-specifically PEGylate TRAIL efficiently and effectively other than N-terminal specific PEGylation.