Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.

The influence of modulating substances on tumor necrosis factor and interleukin-6 levels after injection of murine tumor necrosis factor or lipopolysaccharide in mice.

In this study, we investigated the influence of D-galactosamine (GalN), indomethacin, and dexamethasone on the pharmacokinetics of injected or induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) after a bolus injection of murine TNF (mTNF) or lipopolysaccharide (LPS). It is well known that GalN treatment renders mice much more vulnerable to TNF or LPS lethality. Nevertheless, GalN had no influence on TNF clearance or IL-6 induction after mTNF injection; however, the induced TNF and IL-6 levels were considerably augmented by the GalN cotreatment when a high dose of LPS was injected (GalN was given as a single injection together with TNF or LPS). Indomethacin and dexamethasone, either of which shows a clear protection against TNF/LPS lethality in normal mice, did not change the clearance of injected mTNF, but both reduced the TNF-induced IL-6 levels. Indomethacin did not affect the level and clearance of LPS-induced TNF, whereas the induced IL-6 levels were significantly lower than in the control mice. The circulating TNF and IL-6 concentrations after LPS injection in mice pretreated with dexamethasone were very considerably reduced. Furthermore, neither agent had an influence on the number of TNF binding sites on hepatocytes. We conclude that the strongly enhanced sensitivity of GalN-treated mice towards mTNF-induced or LPS-induced lethality was not reflected in circulating TNF or IL-6 levels, and that dexamethasone and indomethacin both reduce circulating IL-6 concentrations in mice treated with TNF and LPS.

Two discrete types of tumor necrosis factor-resistant cells derived from the same cell line.

From the murine fibrosarcoma cell line L929s, which is sensitive to tumor necrosis factor (TNF)-mediated cell lysis, two discrete types of TNF-resistant variants were derived by TNF selection. Cells of the first type (named L929r1) were not sensitized to TNF cytotoxicity by cotreatment with either inhibitors of protein or RNA synthesis, or gamma-interferon, despite the presence of a functional gamma-interferon response. L929r1 constitutively produced TNF in the supernatant and expressed membrane-bound TNF, which was not bound to the TNF receptor. In fact, TNF receptors could not be demonstrated on L929r1 cells, not even after low pH treatment and/or incubation with antiserum to TNF. L929r1 exhibited a stable TNF-resistant phenotype in the absence of further TNF selection. No evidence could be obtained that TNF acted as an autocrine growth factor for these cells. L929r2, the second type of TNF-resistant L929 cells, became sensitive to TNF lysis in the presence of RNA or protein synthesis inhibitors, or in the presence of gamma-interferon. TNF induced the secretion of interleukin 6 in these cells, additionally showing that functional TNF signaling in these cells indeed takes place, but does not lead to cell lysis under normal conditions. L929r2 did not produce TNF, also not upon stimulation with exogenous TNF. The number and binding affinity of TNF receptors were not consistently different between L929s and L929r2 cells. In the absence of further TNF selection, L929r2 gradually reverted to TNF sensitivity. This sensitivity was not reversible to TNF resistance by the gene-regulatory agents 5-azacytidine or sodium butyrate. Treatment with these agents also did not affect the TNF sensitivity of L929s cells nor the TNF resistance of L929r1 and L929r2 cells. In summary, our results suggest the existence among cells of the same cell line of discrete mechanisms for acquisition of resistance to TNF-mediated cell lysis.

Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.

C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.

Interleukin-6 enhances the expression of tumor necrosis factor receptors on hepatoma cells and hepatocytes.

We have studied the effect of interleukin-6 (IL-6) on the binding of tumor necrosis factor (TNF) to various cell lines. A significant increase (up to 250%) in binding was observed on rat hepatocytes and on the human hepatoma cell line HepG2, while no changes in the number of cells or cell morphology could be observed. Scatchard plot analysis showed that IL-6 enhanced the number of TNF receptors without affecting the receptor affinity. The effect reached plateau levels after approximately 6 h and at IL-6 concentrations of 10 ng/ml. It could be completely eliminated by cotreatment of cells with anti-IL-6 antibodies, but not by treatment with anti-interferon-gamma (IFN-gamma), suggesting that IFN-gamma, which can enhance TNF receptor expression on a variety of cells, was not a mediator in this IL-6 effect. Treatment with inhibitors of protein or RNA synthesis completely abolished the IL-6-induced increase, suggesting that IL-6 caused an enhanced transcription of TNF receptor mRNA. IL-1 had no effect on TNF binding to HepG2. However, when cells were cotreated with IL-1 and IL-6, IL-1 could completely abrogate the IL-6 effect.