Two different cell types have different major receptors for human tumor necrosis factor (TNF alpha)

The receptors for tumor necrosis factor alpha (TNF alpha) were analyzed on myeloid cells (HL60, U937, K562, and freshly isolated blood monocytes) and on cells of epithelial origin (MCF7, HEp2 and HeLa cells), by use of radiolabeled TNF alpha and cross-linking experiments. Both cell types had high but slightly different affinities for TNF alpha. The myeloid cells had major cross-linked products of 98-100 kDa, which were similar in their N-linked glycosylation, whereas the cells of epithelial origin contained a major cross-linked product of 75 kDa, a second product of 95 kDa. The major receptors of both cell types (studied mostly with HL60 and HEp2 cells) are different proteins because (a) their apparent molecular masses were different and no evidence was obtained for cell-specific proteases, which could generate the differently sized receptors from one common receptor molecule; (b) anti-receptor antibodies, which precipitated the 95- and 75-kDa products, did not precipitate the 100-kDa cross-linked complex; (c) the native TNF alpha-receptor complexes had different proteolytic fingerprints; (d) the tryptic fragments differed in their association with the cell membrane vesicles; (e) the receptors differed in their degree of N-linked glycosylation; and (f) O-linked glycosylation was found on the major receptor of HL60 but not of HEp2 cells. In addition, myeloid cells may also contain a small amount of the HEp2-type of TNF alpha receptor. We suggest that at least two different receptors for TNF alpha exist.     

Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells.

HL60 and EL4 cells incubated with tumor necrosis factor-alpha (TNF-alpha) plus staurosporin, a potent inhibitor of protein kinases, showed at least 2-fold increased levels of nuclear factor-kappa B (NF-kappa B) activity compared with TNF-alpha alone both during rapid NF-kappa B activation from the cytosolic pool and protein synthesis-dependent NF-kappa B activation. NF-kappa B activation by phorbol 12-myristate 13-acetate (PMA) and interleukin-1 was inhibited by staurosporin. Staurosporin treatment hardly affected the TNF-alpha-induced increase in mRNA for the p51 subunit of NF-kappa B but interfered with any phorbol ester (PMA)-induced increase in p51 mRNA. Thus, induction of NF-kappa B and p51 mRNA by TNF-alpha was not mediated by a staurosporin-sensitive factor, but NF-kappa B activation by TNF-alpha was even reduced by action of a staurosporin-sensitive factor. Decreased levels of phosphorylation of TNF-R alpha (TNF receptor type alpha) after staurosporin-treatment correlated with increased induction of NF-kappa B by TNF-alpha. Staurosporin-treatment did not affect TNF-R levels. Although protein kinase C stimulation by PMA inhibited NF-kappa B activation by TNF-alpha, its action mechanism may be different from that of the staurosporin-sensitive factor. PMA induced disappearance of TNF-R alpha by shedding into the surrounding medium, with kinetics similar to those of its inhibition of NF-kappa B activation by TNF-alpha. Phosphorylation may not mediate receptor shedding, since PMA treatment did not detectably affect TNF-R alpha phosphorylation.     

Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta.

No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3-methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic deletion of the tumor necrosis factor receptor p60 or p80 abrogates ligand-mediated activation of nuclear factor-kappa B and of mitogen-activated protein kinases in macrophages.

Tumor necrosis factor (TNF) is a pleiotropic cytokine known to regulate cell growth, viral replication, inflammation, immune system functioning, angiogenesis, and tumorigenesis. These effects are mediated through two different receptors, TNFR1 and TNFR2 (also called p60 and p80, respectively), with p60 receptor being expressed on all cell types and p80 receptor only on cells of the immune system and on endothelial cells. Although the role of p60 receptor in TNF signaling is well established, the role of p80 is less clear. In this report, by using macrophages derived from wild-type mice (having both receptors) and mice in which the gene for either p60 (p60(-/-)), or p80 (p80(-/-)), or both (p60(-/-) p80(-/-)) receptor have been deleted, we have redefined the role of these receptors in TNF-induced activation of nuclear factor (NF)-kappa B and of mitogen-activated protein kinases. TNF activated NF-kappa B in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. These results correlated with the I kappa B alpha degradation needed for NF-kappa B activation. We also found that TNF activated c-Jun N-terminal protein kinase in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF activated p38 MAPK and p44/p42 MAPK in wild-type but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF induced the proliferation of wild-type macrophages, but for p60(-/-) and p80(-/-) macrophages proliferation was lower, and in p60(-/-) p80(-/-) it was absent. Overall, our studies suggest that both types of TNF receptors are needed in macrophages for optimum TNF cell signaling.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin).

Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.     

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.     

Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway

We have previously shown that two tumor necrosis factor (TNF) receptors (TNFR) exhibit antagonistic functions during neurodegenerative processes in vivo with TNFR1 aggravating and TNFR2 reducing neuronal cell loss, respectively. To elucidate the neuroprotective signaling pathways of TNFR2, we investigated glutamate-induced excitotoxicity in primary cortical neurons. TNF-expressing neurons from TNF-transgenic mice were found to be strongly protected from glutamate-induced apoptosis. Neurons from wild type and TNFR1(-/-) mice prestimulated with TNF or agonistic TNFR2-specific antibodies were also resistant to excitotoxicity, whereas TNFR2(-/-) neurons died upon glutamate and/or TNF exposures. Both protein kinase B/Akt and nuclear factor-kappa B (NF-kappa B) activation were apparent upon TNF treatment. Both TNFR1 and TNFR2 induced the NF-kappa B pathway, yet with distinguishable kinetics and upstream activating components, TNFR1 only induced transient NF-kappa B activation, whereas TNFR2 facilitated long term phosphatidylinositol 3-kinase-dependent NF-kappa B activation strictly. Glutamate-induced triggering of the ionotropic N-methyl-D-aspartate receptor was required for the enhanced and persistent phosphatidylinositol 3-kinase-dependent NF-kappa B activation by TNFR2, indicating a positive cooperation of TNF and neurotransmitter-induced signal pathways. TNFR2-induced persistent NF-kappa B activity was essential for neuronal survival. Thus, the duration of NF-kappa B activation is a critical determinant for sensitivity toward excitotoxic stress and is dependent on a differential upstream signal pathway usage of the two TNFRs.     

Redox-sensitive transactivation of epidermal growth factor receptor by tumor necrosis factor confers the NF-kappa B activation

Cross-communication between different signaling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. In this paper we have explored the possibility that tumor necrosis factor (TNF) receptor signal cross-talks with epidermal growth factor (EGF) receptor signal on the nuclear factor-kappa B (NF-kappa B) activation pathway. We have demonstrated that overexpression of the EGF receptor (EGFR) in NIH3T3 cells significantly enhances TNF-induced NF-kappa B-dependent luciferase activity even without EGF, that EGF treatment has a synergistic effect on the induction of the reporter activity, and that this enhancement is suppressed by AG1478, EGFR-specific tyrosine kinase inhibitor. We also have shown that TNF induces tyrosine phosphorylation and internalization of the overexpressed EGFR in NIH3T3 cells and the endogenously expressed EGFR in A431 cells and that the transactivation by TNF is suppressed by N-acetyl-l-cysteine or overexpression of an endogenous reducing molecule, thioredoxin, but not by phosphatidylinositol 3-kinase inhibitors and protein kinase C inhibitor. Taken together, this evidence strongly suggests that EGFR transactivation by TNF, which is regulated in a redox-dependent manner, is playing a pivotal role in TNF-induced NF-kappa B activation.     

A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.     

Maintenance of NF-kappa B activity is dependent on protein synthesis and the continuous presence of external stimuli.

The activation of NF-kappa B-like activities (called NF-kappa B) by tumor necrosis factor alpha (TNF alpha) and the phorbol ester phorbol 12-myristate 13-acetate (PMA) were compared. High levels of NF-kappa B activity were found 2 to 4 min after TNF alpha addition to human HL60 cells and lasted for at least 3 h, although the half-life of active NF-kappa B was less than 30 min. Inactive NF-kappa B, however, was relatively stable. NF-kappa B activation by TNF alpha was initially cycloheximide insensitive, but maintenance of NF-kappa B activity required ongoing protein synthesis and continuous stimulation by TNF alpha. Thus, the cells did not remain in an activated state without stimulation. In HL60 cells, NF-kappa B induction by PMA required 30 to 45 min and was completely dependent on de novo protein synthesis, while PMA (and interleukin-1) induced NF-kappa B activity rapidly in mouse 70Z/3 cells via a protein synthesis-independent mechanism. The NF-kappa B-like activities obtained under each condition behaved identically in methylation interference and native proteolytic fingerprinting assays. The NF-kappa B-like factors induced are thus all very similar or identical. We suggest that cell-specific differences in the protein kinase C-dependent activation of NF-kappa B may exist and that TNF alpha and PMA may induce expression of the gene(s) encoding NF-kappa B.     

Induction of tumor necrosis factor-alpha and its receptors during differentiation in myeloid leukemic cells along the monocytic pathway. A possible regulatory mechanism for TNF-alpha production

We examined the characteristics of tumor necrosis factor (TNF) receptors expressed on immature mouse myeloid leukemic cells (M1), M1 cells induced to differentiate into macrophages, and macrophage cells (Mm1 cells) by binding studies with radioiodinated TNF. Scatchard analysis of TNF binding revealed that a single class of high affinity receptor was present and that 750-1,100 receptors were expressed on each immature M1 cell. The number of TNF receptors was increased 1.5-2-fold on differentiated M1 cells and 4-5-fold on Mm1 cells with no change in affinity. The addition of interferon-gamma (IFN-gamma) up-regulated the expression of TNF receptors in differentiated M1 cells and Mm1 cells, while immature M1 cells were insensitive to IFN-gamma. The number of TNF receptors on the differentiated cells was increased 4-5-fold by the treatment with IFN-gamma with no change in the binding constant. The affinity of TNF receptors to human TNF-alpha (Kd = 1.7-2.8 nM) was lower than that to murine TNF-alpha (Kd = 0.2-0.7 nM). The assays for cell growth and [3H]thymidine incorporation suggested that no relation exists between the sensitivity of the cells to TNF-alpha and the number of TNF receptors. Enhancement of TNF-mediated cytotoxicity by the treatment with IFN-gamma did not correlate with increases in the number of TNF receptors. Cytolytic assays using L929 cells demonstrated that the amount of constitutive and lipopolysaccharide (LPS)-induced secretion of TNF-alpha was markedly increased during differentiation. Both the constitutive expression and IFN-gamma-mediated superinduction of TNF receptors, and the constitutive and LPS-induced secretion of TNF-alpha were closely related to the extent of cellular differentiation along the monocytic pathway. The time course of LPS-induced TNF-alpha activity showed a rise-and-decline profile with a peak at 2 h. On the other hand, the time course of the number of cell surface TNF receptors showed a decline-and-rise profile, a mirror image of the TNF-alpha activity time course profile in the supernatant. Anti-TNF-alpha antibody treatment blocked the LPS-induced down-regulation of TNF receptors and increased TNF-alpha mRNA accumulation. We discussed "an autoinhibitory system" in which an internalization of secreted TNF-alpha mediated by its own receptors is involved not only in decreasing TNF-alpha activity in the supernatant but also in reducing TNF-alpha mRNA expression.     

Potentiation of lymphokine-induced macrophage activation by tumor necrosis factor-alpha

In this study, we examined the possible role of TNF-alpha and lymphotoxin (TNF-beta) as cofactors of macrophage activation. The results demonstrate that both TNF were capable of enhancing the cytostatic and cytolytic activity of murine peritoneal macrophages against Eb lymphoma cells. The potentiation of tumor cytotoxicity became apparent when macrophages from DBA/2 mice were suboptimally activated by either a T cell clone-derived macrophage-activating factor or by IFN-gamma plus LPS. Neither TNF-alpha nor TNF-beta could induce tumor cytotoxicity in IFN-gamma-primed macrophages, indicating that TNF cannot replace LPS as a triggering signal of activation. In LPS-resistant C3H/HeJ macrophages, which were unresponsive to IFN-gamma plus LPS, a supplementation with TNF fully restored activation to tumor cytotoxicity. Furthermore, TNF-alpha potentiated a variety of other functions in low-level activated macrophages such as a lactate production and release of cytotoxic factors. At the same time, TNF-alpha produced a further down-regulation of pinocytosis, tumor cell binding and RNA synthesis observed in activated macrophages. These data demonstrate new activities for both TNF-alpha and TNF-beta as helper factors that facilitate macrophage activation. In particular, the macrophage product TNF-alpha may serve as an autocrine signal to potentiate those macrophage functions that were insufficiently activated by lymphokines.