TNF Activation Research Articles

Tumor necrosis factor in congestive heart failure: A mechanism of disease for the new millennium?

Tumor necrosis factor alpha (TNF-alpha), a protein belonging to the family of cytokines, is one of the leading mediators of the immune response to inflammation. Its widespread biological effects are modulated by two circulating binding proteins corresponding to the extracellular domain of the membrane receptors, namely soluble TNF receptors. TNF-alpha was first supposed to be linked with congestive heart failure (CHF) on a cachexia-inducing basis. In patients with advanced CHF, elevated levels of circulating TNF-alpha and soluble TNF receptors have been found. The pathophysiological implications of activation of the TNF system in CHF seem to rely mainly on its effects on the heart and the endothelium. TNF-alpha exerts a negative inotropic effect both directly and indirectly, this latter being mediated by enhancement of nitric oxide production. Moreover, TNF-alpha has been suggested to trigger the apoptotic process in cardiac myocytes. There is consensus on the detrimental role played by TNF-alpha in CHF further supported by the evidence of a temporal association between TNF activation and transition from asymptomatic to symptomatic CHF.

Expression of a negative dominant mutant of human P55 tumor necrosis factor-receptor inhibits TNF and monocyte-induced activation in porcine aortic endothelial cells

Expression of a negative dominant mutant of human P55 tumor necrosis factor-receptor inhibits TNF and monocyte-induced activation in porcine aortic endothelial cells

TNF receptor p55 plays a major role in centrally mediated increases of serum IL-6 and corticosterone after intracerebroventricular injection of TNF.

The aim of this work was to study the relative role of the two TNF receptors (p55 and p75) in the central actions of TNF, studying the elevation of serum corticosterone (CS) and IL-6 levels after injection of recombinant murine (rm)TNF (intracerebroventricularly (i.c.v.)) in normal or p55-deficient (p55 -/-) mice. rmTNF induced high serum IL-6 levels and doubled serum CS in normal mice, whereas no elevation of serum IL-6 or CS was induced in p55 -/- mice. However, a normal CS response was observed in p55 -/- mice after LPS (2.5 microg, i.c.v.). p55 -/- mice also responded, although to a lesser extent than p55 +/+, in terms of LPS-induced IL-6 production. We also injected two agonist Abs specific for the two receptors, alpha p55 and alpha p75. While alpha p55 injected i.c.v. induced a marked elevation in CS and IL-6, alpha p75 induced CS (although less than alpha p55) but no IL-6. rmTNF, which binds both receptors, was more potent in inducing IL-6 and CS than injection of rhTNF, which in mice binds only p55. Finally, we investigated the role of p55 and p75 in IL-6 induction by TNF in a murine brain endothelioma. The results resembled closely those obtained in vivo: rmTNF was more potent than rhTNF and only alpha p55, and not alpha p75, induced IL-6 production. These data indicate that p55 plays a major role in TNF activation of the hypothalamus-pituitary-adrenal axis and in the centrally mediated induction of peripheral IL-6 by TNF, but p75, despite having little IL-6 inductive properties by itself, seems to potentiate p55 induction of IL-6.

TNF Regulates the In Vivo Occupancy of Both Distal and Proximal Regulatory Regions of the MCP-1/JE Gene

In vivo genomic footprinting (IVGF) was used to examine regulatory site occupancy during the activation of the murine inflammatory response gene MCP-1/JE by TNF. In response to TNF, both promoter distal and proximal regulatory regions became occupied in vivo. EMSA analysis showed that while some of the factors involved in expression, including NF-κB, were translocated to the nucleus following TNF treatment, others were already present and able to bind DNA in vitro. Protein kinase inhibitor studies showed that protein phosphorylation was required for TNF activation but not factor assembly. These studies provide evidence for a multistep model of TNF-mediated gene regulation involving chromatin accessibility, transcription factor complex assembly, and protein phosphorylation.

Activation of alveolar macrophage TNF and MCP-1 expression in vivo by a synthetic peptide of C-reactive protein.

Administration of multilamellar vesicles (MLV) encapsulating a synthetic peptide (RS-83277) derived from human C-reactive protein (CRP) augments anti-tumor activity of murine alveolar macrophages and reduces established pulmonary metastases of experimental tumors. To explore mechanisms involved in these phenomena, we investigated cytokine and integrin (CDllb) expression of bronchoalveolar lavage (BAL)-derived alveolar macrophages in control (blank MLV) and RS-83277-MLV-treated C57BI mice. Alveolar macrophage production of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant bioactivity increased at 48 h after treatment with RS-83277-MLV but not control MLV. Chemoattractant activity was neutralized by antibody to monocyte chemoattractant protein-1 (MCP-1), but not irrelevant immunoglobulin G(IgG). Changes were reflected by augmented TNF-alpha and MCP-1 mRNA levels in pulmonary tissue and enhanced CD11b expression on mononuclear leukocytes derived from total lung tissue, but not on BAL-derived alveolar macrophages. Results suggest that RS-83277-MLV treatment is associated with activation of alveolar macrophage TNF-alpha and MCP-1 production and up-regulation of adhesion molecules on pulmonary mononuclear leukocytes but not on alveolar macrophages.

Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24).

Signal transduction pathways involved in apoptotic cell death are poorly understood, although recent studies have implicated sphingomyelin hydrolysis and generation of the second messenger, ceramide. Previous work in this laboratory demonstrated that a serine protease termed AP24 was activated by TNF or UV light and induced DNA fragmentation in isolated nuclei. This study extended these findings to examine the role of these enzymes in apoptosis of the U937 cell line and the mechanism of resistance of its variant, U9-TR. Although this subclone was selected by growth in TNF, it was unexpectedly found to resist apoptosis induced by UV light, but was still sensitive to anti-Fas-induced DNA fragmentation. Here we show that in contrast to normal U937 cells, UV light and TNF both failed to activate neutral or acidic sphingomyelinase or AP24 in the U9-TR variant. However, anti-Fas activated both neutral and acidic sphingomyelinase in the variant comparable to that seen in parental U937. The U9-TR variant could be sensitized to TNF or UV light activation of both sphingomyelinase and DNA fragmentation by the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, exogenous bacterial-derived sphingomyelinase caused U9-TR activation of AP24 and DNA fragmentation comparable to that in the parental U937. Exposure of permeabilized U937 cells to ceramide caused internucleosomal DNA cleavage that was blocked by an inhibitor of AP24. Taken altogether, these findings demonstrate that TNF or UV light activate sphingomyelinase that leads to the generation of ceramide resulting in activation of AP24 and DNA fragmentation in sensitive cells. A selective defect in signals leading to sphingomyelinase activation can confer resistance to apoptosis even though the variant is still sensitive to downstream apoptotic signals such as nuclear DNA fragmentation by activated exogenous AP24.

Activation of the transcription factor NF-KB in GH 3 pituitary cells

Since several genes expressed in the pituitary can bind the transcription factor NF-KB, its presence and regulation was examined in the GH 3 pituitary cell line. An electrophoretic mobility shift assay using nuclear extracts and an oligonucleotide probe corresponding to the Ig KB binding site was employed to identify activated NF-KB. One complex possessed properties characteristic of NF-KB: co-migration with an NF-KB complex and binding specificity restricted to NF-KB binding DNA sequences. Antibodies to the NF-KB subunits NFKBlp50 (p50) and RelA (p65) interacted with the extract-DNA complex. Activation of NF-KB in GH 3 cells was increased by PMA or the cytokine tumor necrosis factor alpha. A synergy between PMA and TNF or a calcium mobilizing agent was seen in NF-KB activation. Further TNF activation was enhanced by TRH. These observations indicate the presence of NF-KB in GH 3 cells and demonstrate its activation by hormones/second messengers that act on pituitary cells.

Inflammatory granuloma formation is mediated by TNF-alpha-inducible intercellular adhesion molecule-1.

Recent studies have demonstrated a crucial role for TNF during inflammatory granuloma formation. In addition, TNF has been shown to up-regulate adhesion molecules that participate in cellular recruitment and lymphocyte activation. In the present study, we have examined the mechanism of TNF activation during Schistosoma mansoni egg granuloma formation and its relationship to the expression of ICAM-1. Our initial studies showed that high affinity human soluble TNFR coupled to the Fc portion of an Ig (sTNFR:Fc construct) could effectively diminish granuloma formation and lymphocyte activation in vivo. We have also assessed the increased expression of ICAM-1, its contribution to granuloma development, and its relationship with TNF during lesion formation. Increased steady state ICAM-1 mRNA expression was observed in primary egg granulomas when compared with normal lung and foreign body (Sephadex bead) granulomas, which suggests a role for ICAM-1 in Ag-induced lesion formation. Subsequent studies have demonstrated that sTNFR:Fc treatment down-regulated granuloma formation and ICAM-1 expression, thus suggesting one mechanism of TNF involvement in granuloma formation was through the induction of ICAM-1. Anti-ICAM-1 decreased the soluble egg Ag-specific T cell proliferation in vitro. In addition, passive immunization of mice with anti-ICAM-1 mAb during primary granuloma formation resulted in an attenuation of lesion development as compared with lesion development in a control Ab-treated group. The proliferative response to soluble egg Ag was also significantly reduced in ex vivo experiments that used spleen cells from the anti-ICAM-1 treated mice. These data demonstrate that both TNF and ICAM-1 participate in lymphocyte activation and granuloma formation and suggest that one mechanism of TNF in granuloma development is through TNF-induced ICAM-1 expression.

Adenovirus genes that modulate the sensitivity of virus‐infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G0 cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A2 (cPLA2), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA2) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.

Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.

Microvascular endothelial cells (EC) recruit circulating leukocytes at sites of inflammation, in part through cytokine-regulated expression of endothelial-leukocyte adhesion molecules. Adhesion molecule expression varies among vascular beds and among EC within microvessels of a particular vascular bed. In the present study, we have examined the patterns of antigen expression and cytokine responsiveness of dermal microvascular endothelial cells (DMEC) in a skin organ culture model and, for comparison, in cell culture. Within the superficial vascular plexus (SVP) of normal skin, CD36 molecule expression is undetectable on capillary loops and is expressed on DMEC in only 20% of the larger, horizontal vessels. CD36 expression is not modulated by cytokines. Endothelial-leukocyte adhesion molecule-1 (ELAM-1) expression induced at 6 and 24 h by TNF or IL-1, is restricted to the venular side of the capillary loop and to the venules proper. Vascular cell adhesion molecule-1 (VCAM-1) expression is not inducible on EC of the SVP in normal skin by TNF, IL-1, or IL-4, alone or in combination at either time point. When inflamed skin is examined in organ culture, SVP EC are cytokine responsive regarding VCAM-1 expression. Within the deep vascular plexus (DVP). CD36 molecules are expressed on EC in all capillaries and small vessels. Both ELAM-1 and, to a lesser extent, VCAM-1 expression are inducible by TNF, IL-1, and/or IL-4 on capillaries and larger microvessels at 6 and 24 h. The larger vessels at the dermal-subcutaneous border were found to be CD36-/ELAM-1+/VCAM-1+ after cytokine treatment. CD36 expression of DMEC in cell culture varies from 47 to 98% of cells (mean 75%) in seven separate isolates and is not modified by cytokines. Upon TNF or IL-1 activation, 50 to 90% of DMEC express ELAM-1 molecules at 6 h and expression persists at high levels for 24 h. VCAM-1 expression is negligible at both times. These results with DMEC differ from human umbilical vein EC analyzed in parallel, which are completely CD36- and show transient ELAM-1 and sustained VCAM-1 expression in response to TNF and IL-1. In summary, we have demonstrated that DMEC comprise a heterogeneous population that differ from umbilical vein EC.

Role of protein phosphorylation in TNF-induced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants.

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4-2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.

Soluble tumor necrosis factor receptor: inhibition of human immunodeficiency virus activation.

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to stimulate human immunodeficiency virus type 1 (HIV-1) replication in both chronically and acutely infected T lymphocytes and monocytes. Transcriptional activation of the HIV long terminal repeat and subsequent increase in virus production are linked to TNF activation of the cellular transcription factor NF-kappa B. Here we report the use of two forms of soluble recombinant type 1 (p80) TNF receptor to inhibit TNF-induced HIV activation in vitro. One receptor form is a monomer containing the entire 236 residues of the extracellular (ligand-binding) portion of p80. A second receptor form is a chimeric homodimer containing these residues fused to a truncated human IgG1 immunoglobulin heavy chain and, thus, resembles a bivalent antibody without light chains. These recombinant receptor proteins were tested for their ability to inhibit TNF-alpha-induced expression of HIV-1 in chronically infected human cell lines. We also examined the ability of the soluble receptors to limit the activation of the HIV-long terminal repeat transcription. The soluble TNF receptor dimer was most effective at blocking TNF-alpha-induced HIV-1 expression in both monocytoid and lymphoid cells. The molar ratio of TNF-receptor dimer to TNF-alpha found to be most effective was, at least, 5:1. We conclude that at specific TNF/soluble TNF-receptor dimer ratios, TNF-alpha-induced HIV-1 transcription and expression can be limited in vitro.

Human T cell leukemia virus type I Tax and phorbol 12-myristate 13-acetate induce expression of the A20 zinc finger protein by distinct mechanisms involving nuclear factor kappa B.

A20 was originally identified as a primary tumor necrosis factor alpha (TNF)-responsive gene, which encodes a 790-amino acid zinc finger protein. A20 is expressed in a wide variety of cell lines, including fibroblasts, in which A20 expression protects cells from TNF cytotoxicity. An analysis of A20 expression in lymphocytic and monocytic cells lines revealed that A20 protein expression correlates with lymphocyte activation and monocyte differentiation. A20 expression was also induced in Jurkat T cells expressing the human T cell leukemia virus type I Tax protein. Transient transfection studies demonstrated that stimulation of A20 transcription by TNF, phorbol 12-myristate 13-acetate (PMA), and Tax was mediated by two kappa B elements within the A20 promoter. Accordingly, DNA electrophoretic mobility shift assays confirmed inducible binding of nuclear factor kappa B (NF-kappa B) to a promoter fragment containing both A20 kappa B elements. Analysis of individual A20 kappa B sites revealed that both kappa B sites were required for TNF or PMA activation of the A20 promoter; however, Tax activation required only one kappa B site. Overexpression of NF-kappa B subunits activated the wild type A20 promoter, but did not activate mutated forms containing single kappa B sites. Thus, Tax activation of A20 transcription occurs through a mechanism distinct from PMA and TNF, possibly due to differential activation of NF-kappa B complexes or transcriptional cofactors.

Neutrophils, monocytes, and lymphocytes bind to cytokine-activated kidney glomerular endothelial cells through L-selectin (LAM-1) in vitro.

The role of L-selectin (LAM-1) as a regulator of leukocyte adhesion to kidney microvascular glomerular endothelial cells was assessed in vitro by using L-selectin-directed mAb and an L-selectin cDNA-transfected cell line. The initial attachment of neutrophils, monocytes, and lymphocytes to TNF-activated bovine glomerular endothelial cells was significantly inhibited by the anti-LAM1-3 mAb. Under static conditions, anti-LAM1-3 mAb inhibited neutrophil adhesion by 15 +/- 5%, whereas the anti-LAM1-10 mAb, directed against a functionally silent epitope of L-selectin, was without effect. The binding of a CD18 mAb inhibited adhesion by 47 +/- 6%. In contrast, when the assays were carried out under nonstatic conditions or at 4 degrees C, the anti-LAM1-3 mAb generated significantly greater inhibition (approximately 60%). CD18-dependent adhesion was minimal (approximately 10%) under these conditions. TNF-activated glomerular endothelial cells also supported adhesion of a mouse pre-B cell line transfected with L-selectin cDNA, but not wild-type cells. This process was also inhibited by the anti-LAM1-3 mAb. Leukocyte adhesion to unstimulated endothelial cells was independent of L-selectin, but, after TNF stimulation, L-selectin-mediated adhesion was observed at 4 h, with maximal induction persisting for 24 to 48 h. Leukocyte adhesion was not observed if glomerular endothelial cells were exposed to TNF in the presence of RNA or protein synthesis inhibitors. Leukocyte attachment to TNF-activated glomerular endothelial cells was also partially inhibited by treatment of the cells with mannose-6-phosphate or phosphomannan monoester, a soluble complex carbohydrate, or by prior treatment of glomerular endothelial cells with neuraminidase, suggesting that the glomerular endothelial cell ligand shares functional characteristics with those expressed by lymph node and large vessel endothelial cells. These data suggest that TNF activation induced the biosynthesis and surface expression of a ligand(s) for L-selectin on glomerular endothelial cells, which supports neutrophil, monocyte, and lymphocyte attachment under nonstatic conditions.

Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor alpha gene

The regulation by tumor necrosis factor alpha (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'-deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5' TGAGCTCA 3', that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.

TNF and IL-1 gene activation in human monocytes treated with krestin (PSK)

TNF and IL-1 gene activation in human monocytes treated with krestin (PSK)

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)