Effects of tumor necrosis factor (TNF) on transplanted tumors induced by methylcholanthrene in mice. A histopathologic study

The effects of a highly purified tumor necrosis factor (TNF) on transplanted methylcholanthrene (Meth A)-induced murine tumors were compared with those of lipopolysaccharide (LPS). TNF caused immediate subepidermal edema and hyperemia followed 2 h later by fibrin thrombi in tumor blood vessels. Finally hemorrhagic necrosis with dispersal of tumor cells occurred. LPS produced similar hemorrhagic necrotizing changes. However, the necrotic action of LPS was delayed and complete tumor regression was not achieved with LPS. These findings suggest that tumor necrosis induced by TNF is due to circulatory disturbance associated with a microvascular injury in the tumor manifested by hyperemia and multiple fibrin thrombi.     

Tumor necrosis factor stimulates gelatinase and collagenase production by granulation tissue in culture

Tumor necrosis factor (TNF, 2.6 X 10(-11)-10(-9) M) caused an increase in the production of active gelatinase and latent collagenase by granulation tissue in culture. As determined by SDS-substrate polyacrylamide gel electrophoresis, granulation tissue produced mainly two species of gelatinase with molecular weights of 64 kDa and 57 kDa, and an additional 80-kDa gelatinase was produced by TNF treatment. The results suggest that TNF may play a role in a rapid collagen turnover in the carrageenin-induced granulation tissue in rats.     

Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

Cytological features of the development of multi-organ failure induced by exposure of the body to tumor necrosis factor]

Morphologic analysis of the changes in the internal organs after i.v. injection of 20 micrograms per mice of tumor necrosis factor (TNF) revealed the development of signs of multiorgan failure (interstitial and intraalveolar edema in the lung, acute fatty liver, tubular necrosis in kidney, brain edema) during first 24 h. The microvascular endothelial cells played a particular role in the state development. Results of investigation proved that endothelial cells were one of the major cellular targets for TNF action in vivo.     

Influence of TNF-alpha and IL-6 infusions on insulin sensitivity and expression of IL-18 in humans

Inflammation is associated with insulin resistance, and both tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 may affect glucose uptake. TNF induces insulin resistance, whereas the role of IL-6 is controversial. High plasma levels of IL-18 are associated with insulin resistance in epidemiological studies. We investigated the effects of TNF and IL-6 on IL-18 gene expression in skeletal muscle and adipose tissue. Nine human volunteers underwent three consecutive interventions, receiving an infusion of recombinant human (rh)IL-6, rhTNF, and saline. Insulin sensitivity was assessed by measurement of whole body glucose uptake with the stable isotope tracer method during a euglycemic hyperinsulinemic clamp (20 mU.min(-1).kg(-1)), which was initiated 1 h after the IL-6-TNF-saline infusion. Cytokine responses were measured in plasma, muscle, and fat biopsies. Plasma concentrations of TNF and IL-6 increased 10- and 38-fold, respectively, during the cytokine infusions. Whole body insulin-mediated glucose uptake was significantly reduced during TNF infusion but remained unchanged during IL-6 infusion. TNF induced IL-18 gene expression in muscle tissue, but not in adipose tissue, whereas IL-6 infusion had no effect on IL-18 gene expression in either tissue. We conclude that TNF-induced insulin resistance of whole body glucose uptake is associated with increased IL-18 gene expression in muscle tissue, indicating that TNF and IL-18 interact, and both may have important regulatory roles in the pathogenesis of insulin resistance.     

Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells

Human recombinant tumor necrosis factor-alpha (TNF) was found to stimulate the production of prostaglandins (PG) by cultured rat mesangial cells. This effect was demonstrable from 6 h, was dose dependent and affected the synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. It required both RNA and protein synthesis but was not associated with a modification of cell proliferation. TNF also stimulated adenosine 3'-5' cyclic monophosphate (cAMP) levels in the mesangial cell culture medium. Indomethacin suppressed the effect of TNF on PGs but only reduced that on cAMP, indicating that PG production partly mediates the increase in cAMP. These findings demonstrate that mesangial cells can be a target for TNF and that the mechanism of TNF action includes stimulation of both PG production and cAMP levels.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

Early response to tumor necrosis factor of human promyelocytic leukemia cell lines sensitive and resistant to the factor

Early cellular events with respect to protein synthesis and the steady-state level of cellular myc (c-myc) mRNA were analyzed in the tumor necrosis factor (TNF)-sensitive human promyelocytic leukemia cell line HL-60 and in its TNF-resistant variant HL-60R after their exposure to TNF. Addition of TNF at 100 units (U)/ml induced de novo synthesis of two proteins with apparent molecular masses of 100 kDa and 40 kDa in HL-60 cells. The induced synthesis of the 100 kDa protein continued for 6 h, while that of the 40 kDa protein was transient. The 100 kDa protein was detectable in HL-60R cells which were maintained in medium containing 1,000 U/ml TNF, whereas the synthesis of the 40 kDa protein could be transiently induced by TNF at 10(5) U/ml. Dot blot hybridization revealed that the steady-state level of c-myc mRNA in HL-60 cells was transiently reduced by TNF at 100 U/ml but remained at a reduced level for 6 h when 10(5) U/ml TNF was present. In HL-60R cells, TNF at 10(5) U/ml could transiently reduce the c-myc mRNA level. These results showed that induction of the synthesis of a 40 kDa protein and a reduction in the steady-state level of c-myc mRNA were concomitant with cellular sensitivity to the cytostatic action of TNF in HL-60 cells.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

Rapid acquisition of an enhanced capacity to produce tumor necrosis factor, alpha/beta interferon, and interleukin 6 after implantation of tumor cells.

This study shows that the ability of mice to produce tumor necrosis factor (TNF), alpha/beta interferon (IFN-alpha/beta), and interleukin 6 (IL-6), but not interleukin 1 (IL-1), in response to endotoxin was dramatically augmented within 24 h of intradermal implantation of 10(6) tumor cells. Tumor cell implantation also caused endotoxin-independent appearance of IFN-alpha/beta and IL-6 in serum within 24 h. Priming for endotoxin-induced TNF production was not evident during the first 12 h of tumor cell implantation and it had decreased by 72 h. However, this decrease was followed by a second peak of priming on day 6 of tumor growth. Priming for endotoxin-induced TNF production was not induced by injection of dead tumor cells, the products of live tumor cells, or syngeneic or allogeneic splenocytes. Priming for TNF production was associated with an increased susceptibility of mice to endotoxin toxicity. These data suggest the existence of a cytokine-dependent host defense mechanism that is rapidly elicited in response to tumor cell implantation.     

Dexamethasone inhibits the cytotoxic activity of tumor necrosis factor

Effect of dexamethasone (DEX) on the cytotoxic activity of tumor necrosis factor (TNF) was examined using murine fibroblast cell line (L929 cells). DEX protected cells from the cytotoxic action of TNF. Protection of cytotoxic action was apparent when cells were pre-treated with DEX for 12h and no protection was observed in the presence of cycloheximide. These results suggested that de novo synthesis of new proteins was required for DEX-mediated protection. Moreover, prolonged simultaneous treatment with TNF and DEX resulted in the enhancement of cell growth, suggesting that TNF acted as a growth factor when cells were protected from the cytotoxic action of TNF. These results suggested that the signal transduction system for fibroblast growth enhancing and cytotoxic action of TNF were different from each other and that the interaction between TNF and glucocorticoids may play a modulating role in some inflammatory processes in vivo.     

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.     

Tumor necrosis factor production and accumulation of inflammatory cells in the corpus luteum of pseudopregnancy and pregnancy in rabbits

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.     

Immunohistochemical evidence for the production of tumor necrosis factor by murine MH134 tumor cells as well as monocytes in tumor lesions after systemic administration of a polyalcoholized mannoglucan from Microellobosporia grisea

The endogenous production of tumor necrosis factor (TNF) by tumor tissues was examined. MH134 tumor cells as well as monocytes in MH134 tumor tissues produced TNF after systemic administration of a polyalcoholized mannoglucan (MGA) from Microellobrosporia grisea as shown by the indirect immunofluorescence technique. MH134 tumor cells also produced TNF when stimulated with lipopolysaccharide in vitro. These results suggest that regression of MH134 hepatoma can be ascribed to TNF produced not only by monocytes but also by tumor cells themselves in the tumor tissue.     

Effect of RU 38486 on TNF production and toxicity.

Glucocorticoid steroids provide considerable protection against the systemic toxicity of tumor necrosis factor-alpha (TNF-alpha, cachexin). In animal experiments RU 38486 (mifepristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone-treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen of lipopolysaccharide (LPS)-treated animals. In tissue cultures RU 38486 induced the TNF synthesis of myeloid cells and increased the TNF production of genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.     

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.     

Early inflammatory cytokine and acute phase protein response under the stress of thermal injury in rats

The acute inflammatory response associated with thermal injury was examined in rats. The appearance of mediators of inflammation in the systemic circulation, including cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6 (IL-6) and acute phase proteins were assessed during initial 72 h following thermal injury. Increased levels of activity were noted for all three cytokines, but with a different time-course. While serum IL-1 activity was elevated throughout the 3-day period of observation, the levels of serum TNF activity were enhanced after 12 h and on days 1 and 3 following scalding injury. The values of IL-6 were already increased one hour after thermal injury and increased progressively up to day 1 following scalding. Alpha2-macroglobulin and haptoglobin levels were increased 12 h after thermal injury, rising further on days 1 and 3. Positive correlation was found between the time-course of increased serum IL-6 activity and alpha2-macroglobulin, as well as between TNF and haptoglobin in the serum.     

Tumor necrosis factor regulates tyrosine phosphorylation on epidermal growth factor receptors in A431 carcinoma cells: evidence for a distinct mechanism.

Previous studies of tumor necrosis factor (TNF) action on tumor cells revealed a possible role for tyrosine phosphorylation of epidermal growth factor (EGF) receptor in the growth-regulatory activities of this cytokine (N. J. Donato, G. E. Gallick, P. A. Steck, and M. G. Rosenblum, J. Biol. Chem., 264: 20474-20481, 1989). EGF receptor immunoprecipitated from [32P] phosphate-equilibrated A431 cells demonstrated that TNF treatment resulted in both a time- and concentration-dependent stimulation of EGF receptor phosphorylation, which was maximal (approximately 3-fold) after 10-20 min of TNF exposure (10 nM). Incubation of A431 cells with an equivalent concentration of EGF resulted in similar stimulation of EGF receptor phosphorylation, albeit at different phosphotyrosine levels. Antiphosphotyrosine immunoblot analysis confirmed these results but suggested that the extent and kinetics of TNF-induced tyrosine phosphorylation were distinct from those obtained in EGF-treated cells. Resolution of tryptic phosphopeptides from EGF receptor demonstrated that TNF-induced phosphorylation of EGF receptor was similar, but not identical, to profiles obtained from EGF-treated cells and distinct when compared to the actions of phorbol ester. Unlike EGF, TNF was unable to directly stimulate EGF receptor tyrosine kinase activity in membranes prepared from A431 cells. In addition, TNF treatment had no significant effect on either the high- or low-affinity ligand-binding sites on EGF receptor and did not alter the kinetics or extent of ligand-induced internalization of EGF receptors. However, EGF receptor biosynthesis was consistently increased upon prolonged treatment with TNF (4-12 h). Our results suggest that TNF regulates both phosphorylation and biosynthesis of EGF receptor in a manner distinct from that of both EGF and phorbol ester, and studies of the differential phosphorylation of EGF receptor may aid in understanding the molecular mode of TNF action.