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)     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.     

Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.     

Tumor necrosis factor-mediated cytotoxicity involves ADP-ribosylation

The mechanism of TNF-mediated cytotoxicity was studied in several cell lines, including L929 murine fibroblasts. TNF caused a time- and dose-dependent increase of ADP-ribosylation in L929 target cells parallel to cell death. During the course of TNF-mediated cytotoxicity in the presence of actinomycin D, an increase in ADP-ribosylation became apparent between 4 and 6 h after exposure to TNF. Intracellular NAD+ and ATP levels decreased parallel to but not preceding cell death. Two inhibitors of ADP-ribosylation, namely 3-aminobenzamide and nicotinamide, prevented TNF-mediated cytotoxicity. Another target, the human cervical carcinoma cell line ME-180, showed an increase in ADP-ribosylation when treated with TNF, and the cytotoxic action of TNF on this target cell was inhibited by these two inhibitors. In the absence of actinomycin D, treatment of L929 cells with TNF also increased ADP-ribosylation, and the cytotoxic action of TNF was inhibited by nicotinamide. These results indicate that ADP-ribosylation may be involved in the TNF-mediated cytotoxic reaction.     

Cytotoxicity mediated by tumor necrosis factor in variant subclones of the ME-180 cervical carcinoma line: modulation by specific inhibitors of DNA topoisomerase II

The mechanism of tumor necrosis factor (TNF)-induced cytotoxicity has been investigated using two clonal variants of the ME-180 human cervical carcinoma cell line. The clonal lines were characterized with respect to their expression of TNF receptors, kinetics of cell death, and their ability to communicate intercellularly through gap junctions. The ME-180.4 and ME-180.8 clones were identified by their relative sensitivity to TNF induced lysis in a 24-h assay. The dose of TNF required to kill 50% of the target cells was 60 pM for the sensitive ME-180.4 and 2.5 nM for the ME-180.8. However, when assay times were extended, the dose response for both clones was the same, indicating that a difference in the kinetics of cell death and not absolute TNF sensitivity existed between the ME-180.4 and ME-180.8 clones. Both clones were gap junction deficient as judged by their inability to transfer Lucifer yellow or 6-carboxyfluorescein, a characteristic phenotype of cells sensitive to cytotoxicity by TNF. The level of surface receptor expressed on these clones was nearly identical with a Kd = 0.3 nM and 5,000 binding sites per cell. Measurement of the kinetics of cell death revealed that the time between the addition of TNF and the onset of observed cell death (induction phase) was much shorter for the ME-180.4 (32-55 h) than for the resistant ME-180.8 (55-80 h). Mitomycin C, a DNA alkylating agent, significantly reduced the length of the induction phase for both clones, although the kinetic difference between the clones remained unchanged. Two epipodophyllotoxins, VP-16 and VM-26, which specifically inhibit the rejoining activity of DNA topoisomerase II, showed a 10-100-fold synergistic effect when combined with TNF as shown by isobologram analysis. VM-26 when added to the resistant ME-180.8 clones decreased the length of induction phase and abolished the kinetic difference observed with the ME-180.4 clone. These results indicate that the variance in the TNF response of these two clones was closely associated with DNA topoisomerase II, and suggest that this enzyme may play an important role in TNF mediated cytotoxicity.     

Dialysable leucocyte extract (DLE) reduces lipopolysaccharide- induced tumour necrosis factor secretion in human leucocytes

Dialysable leucocyte extract (DLE), obtained from lysed leucocytes, provide clinical effectiveness in a broad spectrum of diseases. Tumour necrosis factor (TNF) is raised in AIDS patients leading to an increase in human immunodeficiency virus (HIV) replication in vitro [1,2], whereas progression to AIDS in asymptomatic HIV infected individuals is retarded under treatment with DLE. In the present study we tested the DLE effect in vitro on both TNF biological activity (cytotoxicity) in L929 cells and its induction by lipopolysaccharide (LPS) in human monocytes as well as in whole blood from healthy donors. When monocytic cells were simultaneously exposed to LPS and DLE during a period of 5 1/2 hours, the induction of TNF was strongly diminished. The same inhibitory effect of DLE on TNF induction was observed when LPS was added to the culture medium prior to DLE. No significant effect of DLE on TNF-mediated cytotoxicity, even in the presence of the highest concentrations of DLE tested, was detected. DLE treatment of whole human blood regulates responses to LPS: simultaneous in vitro exposure to endotoxin provokes a remarkable decrease (4- and 1.6-fold) of TNF release. In pre-incubation experiments, TNF production was largely reduced or completed abrogated. These results could, in part, explain the in vivo observed effect, when under treatment with this extract, the progression to AIDS of HIV-infected individuals was retarded. The results suggest that ‘natural’ substances like DLE may be important immunomodulators in inflammatory diseases.     

Effect of tumor necrosis factor/cachectin on the activity of the low density lipoprotein receptor on human skin fibroblasts

We have investigated the effects of recombinant human tumor necrosis factor (TNF)/cachectin on the cellular binding of human low density lipoprotein (LDL) to human skin fibroblasts. When recombinant TNF was added to cultured cells, LDL binding doubled after 24 h of incubation. The effect of TNF was dose-dependent and its maximal effect was observed at concentrations of 1-10 ng/ml. TNF also stimulated the growth of human skin fibroblasts 1.6-fold. These results indicate that TNF increases LDL-receptor activity, which might be related to its stimulatory effect on cell growth.     

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.     

Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor

Tumor necrosis factor (TNF) induces the synthesis of protein(s) that can protect cells against subsequent killing by TNF in the presence of cycloheximide. Here we demonstrate that manganous superoxide dismutase (MnSOD), a mitochondrial enzyme involved in the scavenging of superoxide radicals (O2-), is such a protein. Overexpression of MnSOD confers increased resistance to TNF plus cycloheximide on the 293 human embryonic kidney cell line. Conversely, expression of antisense MnSOD RNA renders these cells sensitive to TNF even in the absence of cycloheximide. The TNF sensitivity of the ME-180 human cervical carcinoma cell line can also be modulated through expression of sense and antisense MnSOD RNAs. These data identify MnSOD as an important determinant of cellular resistance to TNF and implicate mitochondrially generated O2- as a key component of TNF-mediated tumor cell killing.     

Use of a sensitive receptor binding assay to discriminate between full-length and truncated human recombinant tumor necrosis factor proteins

A radioreceptor assay (RRA) capable of detecting picomolar concentrations of human recombinant tumor necrosis factor (TNF) was used to compare the relative binding affinities of genetically engineered full-length and truncated TNF proteins. The specific cell-surface receptors for TNF present on the human cervical carcinoma cell line ME-180 were characterized as having a Kd of 0.2 nM and a density of 2700 sites/cell. Conditions were then defined for an RRA that maximized the specific binding of 125I-TNF to this adherent cell line. Incubation of ME-180 cells with 125I-TNF at 37 degrees C in the presence of 0.02% sodium azide resulted in a 4-fold increase in assay sensitivity and a doubling of specific counts bound, as compared to binding done at 4 degrees C with or without sodium azide. Inhibition of receptor-ligand internalization under these conditions was a likely reason for the increases. This system was utilized to compare low concentrations of the full-length TNF protein and a genetically altered TNF protein (mutein) which lacks the 10 N-terminal amino acids and contains an N-terminal methionine. Previous studies showing the truncated TNF to be 2- to 3-fold lower in cytotoxic activity on a variety of tumor cell lines were corroborated by our findings that the mutein was also three and one-half times lower in relative affinity for the TNF receptor on ME-180 cells. These results suggest a possible role for these residues in receptor binding and illustrate the use of a highly sensitive RRA for the evaluation of TNF molecules altered by recombinant DNA technology.     

Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity.

Heat treatment and various other stresses render tumor cells resistant to cytotoxicity mediated by tumor necrosis factors (TNFs). Here, we elucidate the molecular basis of this phenomenon by demonstrating that the major heat shock protein, hsp70, protects tumor cells from TNF cytotoxicity even in the absence of stress. The human hsp70 gene was stably introduced into highly TNF-sensitive WEHI-S tumor cells both in the sense and antisense orientation. All clones constitutively expressing the exogenous human hsp70 gene were protected from TNF-mediated killing approximately 1000-fold. Remarkably, the growth of one clone was actually stimulated by low concentrations of TNF. Moreover, a clone expressing antisense hsp70 RNA was rendered extremely sensitive to TNFs. Hsp70-mediated protection from TNF cytotoxicity was confirmed in transient expression experiments employing retroviral vectors. Changes in cellular sensitivity to TNF were not associated with alterations in the binding of TNF to its receptors. Neither the transfection procedure itself nor overexpression of the low molecular weight heat shock protein, hsp27, had any effect on cellular susceptibility to TNFs. Our data suggest that hsp70 may increase the oncogenic potential of some tumor cells by providing them with an escape mechanism from immunological defense.     

Hematopoietic progenitor cells of transgenic mice with increased copper/zinc-superoxide dismutase activity are resistant to tumor necrosis factor

The mechanism of growth inhibition mediated by tumor necrosis factor (TNF) is unclear. Since recent data strongly suggested that generation of superoxide is a key step in cytotoxicity of TNF, we reasoned that cells expressing high levels of enzymes that degrade superoxide radicals would be resistant to TNF. Therefore, we examined the TNF-sensitivity of bone marrow progenitor cells of transgenic mice that expressed the gene for human copper zinc-superoxide dismutase (CuZn-SOD). The CuZn-SOD is a key enzyme in the metabolism of superoxide radicals. Heterozygous and homozygous transgenic mice had 3- and 5-fold increased levels of CuZn-SOD activity, respectively. Bone marrow cells of transgenic and nontransgenic mice were plated in soft gel culture with TNF (0.01–100 ng/ml). TNF inhibited myeloid colony formation supported by either granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF from nontransgenic mice in a dose-dependent manner. In contrast, the myeloid clonal growth of homozygote transgenic mice was not inhibited by TNF at concentrations up to 100 ng/ml. As expected, the effects of TNF on erythroid clonogenic cells, which do not produce superoxide, and the action of transforming growth factor-β on myeloid progenitor cells, were similar in both transgenic and nontransgenic mice. These results suggest that the mechanism of TNF-mediated growth inhibition of hematopoietic cells occurs through production of superoxide. © 1994 Wiley-Liss, Inc.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Stimulation of ornithine decarboxylase activity in chick fibroblasts by non-suppressible insulin-like activity (NSILA), insulin and serum.

A factor isolated from human serum (nonsuppressible insulin-like activity, NSILA) stimulates multiplication of serum-starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 muU/ml) and pharmacological doses of insulin (200 mU/ml) stimulate ODC 4-5-fold, 10% fetal calf serum about 18-fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity. A sharp increase in ODC activity occurs between 2, 5 and 5.0 hours after addition of the growth factors with a peak at 4.0-4.5 hours ("activation period"). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire "activation period." The potential to reactivate ODC decreases as cells pass through S phase. Results obtained using cycloheximide suggest that ODC is translated only in the second half of the "activation period." Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.     

Tumor necrosis factor and IFN induce a common set of proteins

The treatment of cells with TNF or IFN results in the development of an antiviral state and in the induction of a common set of proteins with m.w. of 80,000, 67,000, and 56,000. The induction of the 80,000- and 56,000-Da proteins after TNF treatment is dependent on the synthesis of an intermediary protein, whereas the induction of the 67,000-Da protein appears to occur as a direct result of the TNF treatment. The effects of antibodies to IFN on the TNF-mediated effects have been evaluated and reveal that the incubation of TNF-treated cells with antibody to rIFN-beta 1 greatly reduces the antiviral effectiveness of the TNF treatment and blocks the ability of TNF to induce the 80,000-Da protein. Incubation with antibodies to either IFN-alpha or IFN-gamma failed to affect the TNF-mediated responses. Thus, the induction by TNF of each of the proteins is regulated differently and is mediated through both IFN-dependent and IFN-independent mechanisms.     

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.