Tumor necrosis factor inhibits human myogenesis in vitro.

We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.     

Evidence for the existence of two forms of membrane tumor necrosis factor: an integral protein and a molecule attached to its receptor

Plasma membranes were isolated from thioglycolate-induced peritoneal mouse macrophages and tested directly in a 51Cr-release assay against WEHI 164 tumor cells. These membranes showed anti-TNF antibody inhibitable killing of the TNF-sensitive tumor cell line, indicating that membrane-associated TNF is present on mouse macrophages. In order to elucidate whether membrane TNF is an integral protein or a molecule attached to a receptor, cells and plasma membranes were treated with low pH buffer. A partial reduction in TNF activity was observed which could be restored by incubation with exogenous TNF. In a Western blot analysis the integral membrane TNF could be identified as the 26-kDa molecule on activated mouse macrophages. These results indicate that both forms of membrane-associated TNF exist on macrophages and are responsible for cell-mediated cytotoxicity against TNF-alpha-sensitive targets.     

Murine tumor cells transduced with the gene for tumor necrosis factor-alpha. Evidence for paracrine immune effects of tumor necrosis factor against tumors

Studies of the anti-tumor activity of TNF-alpha in vivo have been hampered by the need to administer systemically toxic doses of the cytokine to obtain a curative response. To facilitate studies of the effect of high local concentrations of TNF-alpha on tumor growth and host immunity, a newly induced murine sarcoma was transduced with the gene for human TNF-alpha and the biologic characteristics of these cells were examined. We identified high and low TNF-producing tumor clones which exhibited stable TNF secretion over time. Significant amounts of membrane associated TNF were found in a high-TNF producing clone as well. No difference in the in vitro growth rates between TNF-producing and nonproducing cell lines was observed. In contrast, in vivo studies demonstrate that although unmodified parental tumor cells grew progressively when implanted s.c. in animals, tumor cells transduced with the TNF gene were found to regress in a significant number of animals after an initial phase of growth. This effect correlated with the amount of TNF produced and could be blocked with a specific anti-TNF antibody. Regressions of TNF-producing cells occurred in the absence of any demonstrable toxicity in the animals bearing these tumors. TNF-producing tumor cells could function in a paracrine fashion by inhibiting the growth of unmodified, parental tumor cells implanted at the same site. The ability of tumor cells to regress was abrogated by in vivo depletion of CD4+ or CD8+ T cell subsets and animals that had experienced regression of TNF-producing tumors rejected subsequent challenges of parental tumor. Our studies thus show that tumor cells elaborating high local concentrations of TNF regress in the absence of toxicity in the host and that this process requires the existence of intact host immunity. Studies of the lymphocytes infiltrating the gene modified tumors and attempts to use TNF gene modified tumor infiltrating lymphocytes to deliver high local concentrations of TNF to the tumor site without inducing systemic toxicity are underway.     

Genetic construction and characterization of a fusion protein consisting of a chimeric F(ab') with specificity for carcinomas and human IL-2.

A genetic construct was created incorporating gene fragments encoding the H chain V region of the human carcinoma specific antibody L6, the CH1 domain of human IgG1, a linker region, and human IL-2. This construct was cotransfected with a chimeric L6 L chain construct into the murine myeloma cell line Ag8.653 for expression. First round clones produced the fusion protein at an estimated 5 to 10 micrograms/ml based on idiotypic reactivity. Dual binding activity was demonstrated through specific interaction with the L6 Ag on human tumor cells and the IL-2R on activated human T cells. The IL-2 portion of the molecule was shown to support the growth of the IL-2-dependent T cell line CTLL2, and the qualitative nature of the IL-2 signal was found to be the same as rIL-2 with respect to induction of tyrosine-phosphorylation of intracellular protein substrates. Tumor cells coated with the fusion protein were shown to cause T cell proliferation and the presence of the fusion protein was found to enhance cell-mediated destruction of human tumor cells.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor

Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.     

ATROSAB,a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through TNFR1 and TNFR2, two membrane receptors, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB produced in CHO cells showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1–70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.Key words: humanized IgG, antagonistic antibody, tumor necrosis factor receptor 1, epitope mapping     

Co-migration and internalization of transferrin and its receptor on K562 cells

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.     

Mitogenic action of tumor necrosis factor in human fibroblasts: interaction with epidermal growth factor and platelet-derived growth factor

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS-4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS-4 fibroblasts in a chemically defined, serum-free medium in the absence of EGF. Anti-EGF receptor antibody, which blocked the mitogenic effects of EGF in FS-4 cells, did not inhibit the mitogenic action of TNF in serum-free or serum-containing medium, indicating that EGF or an EGF-like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum-free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth-arrested FS-4 cells and were compared with the action of platelet-derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth-arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth-arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth-arrested FS-4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.     

In vivo tumor delivery of a recombinant single chain Fv::tumor necrosis factor-alpha fusion [correction of factor: a fusion] protein.

Locoregional and intratumoral administration of tumor necrosis factor alpha (TNF alpha) has been successful in obtaining inhibition or regression of tumor growth in the clinic. This potent antitumor activity of TNF alpha has not yet been exploited as a systemic agent in cancer therapy, mainly due to high levels of toxicity to normal tissues before a therapeutic dose of TNF alpha in the tumor has been achieved. To address this, we have targeted TNF alpha using antitumor antibodies. We have used a genetic fusion of human recombinant TNF alpha with MFE-23, a single-chain Fv antibody fragment directed against carcinoembryonic antigen. MFE-23::TNF alpha fusion protein is isolated in high yields (28 mg/L) from bacterial inclusion bodies and purified to homogeneity by affinity chromatography. It is a 144 kDa trimer in native form and possesses the antigen-binding activity of the sFv and the cytotoxicity to both WEHI 164 and a human adenocarcinoma cell line (LoVo) of rhTNF alpha. Radiolabeled MFE-23::TNF alpha binds both human and mouse TNF receptor 1 in vitro and is able to localize effectively in nude (nu/nu) mice bearing human LS174T xenografts; tumor/tissue ratios of 21:1 and 60:1 are achieved 24 and 48 h after intravenous injection. These studies indicate that MFE-23::TNF alpha will provide an effective means for systemically administered cancer therapy with TNF alpha.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells

Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.     

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.     

Morphology and lytic mechanisms of interleukin 3-dependent natural cytotoxic cells: tumor necrosis factor as a possible mediator

Populations of interleukin 3 (IL 3)-dependent cells can be derived from mouse bone marrow that display natural cytotoxicity (NC) against Wehi-164 target cells but do not display natural killing against YAC-1 cells. These bone marrow-derived NC cells cultured up to 2 mo in IL 3 do not contain rearranged T cell receptor beta-chain genes. They appear to be mast-like cells by electron microscopy and contain heterogeneous type granules. The molecules that mediate NC appear to be contained in these granules and are preformed because protein synthesis inhibitors have no effect on the capacity of IL 3-dependent NC cells to lyse Wehi-164 target cells. In addition to the IL 3-dependent bone marrow-derived cells, the basophilic leukemia cells, RBL-1, but not P815 mastocytoma cells were found to mediate NC against Wehi-164 cells. Both bone marrow-derived NC and RBL-1 cells can lyse L929 cells in 18 hr, suggesting that the putative NC mediator may be related to lymphotoxin/tumor necrosis factor (TNF). Recombinant human TNF displayed identical properties as NC cells; both entities possessed the same target cell specificity and had similar kinetics of target cell killing. The use of polyclonal rabbit antimouse TNF antibody blocked the actions of NC cells. Thus we believe that the mediation of NC is through the actions of a TNF-like molecule.     

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis is mediated by tumor necrosis factor alpha in human monocytic U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-alpha (TNFalpha) in U937 cells, and exogenously added TNFalpha induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFalpha antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFalpha and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFalpha in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFalpha is an autocrine-stimulating factor for the induction of apoptosis in these cells.