Faculty Bibliography

Tumor necrosis factor (TNF), interleukin-1 (IL-1), and epidermal growth factor (EGF) were mitogenic for human diploid FS-4 fibroblasts. Dexamethasone amplified the growth-stimulating action of all three agents. Amplification of the growth-stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS-4 cells. TNF and IL-1 increased the steady state level of interferon (IFN)-beta 2 mRNA but failed to induce detectable levels of IFN-beta 1 mRNA in FS-4 cells. Dexamethasone inhibited the increase of IFN-beta 2 mRNA levels by IL-1 or TNF. Inhibition of IFN-beta synthesis is likely to be responsible for the inhibition of the TNF-induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN-beta synthesis may also be responsible for the amplification by dexamethasone of the growth-stimulating action of TNF and IL-1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by different mechanism

Highly purified recombinant human tumor necrosis factor (TNF) was found to induce interleukin 1 (IL 1) production in diploid human FS-4 fibroblasts. Demonstration of IL 1 activity was based on the ability of TNF-treated FS-4 cells, subsequently fixed with formaldehyde, to stimulate thymocyte proliferation in the presence of phytohemagglutinin. Incubation of FS-4 cells with the optimal dose of TNF (10 ng/ml) resulted in a marked increase in [3H] thymidine uptake by thymocytes co-cultured with formaldehyde-fixed FS-4 cells. Induction of this apparently membrane-associated IL 1 (MA-IL 1) activity was demonstrable at 6 hr and reached a plateau after 48 hr of incubation with TNF. FS-4 cells did not secrete soluble IL 1 in response to TNF. Dexamethasone suppressed the synthesis of TNF-induced MA-IL 1. A monoclonal antibody specific for TNF neutralized MA-IL 1 induction, indicating that the induction is due to TNF, and not to a contaminant in the TNF preparation. The ability of TNF to induce IL 1 synthesis in FS-4 fibroblasts at the transcriptional level was confirmed by S1 nuclease protection assay. Cytoplasmic RNA from uninduced FS-4 cells contained no demonstrable RNA hybridizing with a human IL 1-alpha cDNA probe and low levels of RNA hybridizing with an IL 1-beta cDNA. Induction with TNF resulted in the appearance of IL 1-alpha mRNA and a very significant increase in IL 1-beta mRNA, indicating that TNF induces the synthesis of both IL 1-alpha and IL 1-beta in FS-4 cells

Biological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L929 cells, one derivative, designated as TNF(Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L929 cells, suggesting that the cytotoxic activity of TNFs on L929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF(Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not

Tumor necrosis factor (TNF) was shown previously to stimulate the growth of human FS-4 fibroblasts. Here we show that human recombinant TNF can increase the binding of epidermal growth factor (EGF) to these cells. Incubation with TNF resulted in a 40-80% increase in the number of EGF-binding sites with no apparent change in receptor binding affinity. The increase in EGF binding was apparent 8-12 h after the addition of TNF. TNF also increased the amount of EGF receptor protein immunoprecipitated from cells labeled with [35S]methionine. Stimulation of EGF receptor protein synthesis was demonstrable 2-4 h following TNF treatment. TNF increased EGF binding with a dose-response relationship similar to that reported earlier for the mitogenic action. Increased expression of EGF receptors, due to enhanced synthesis of the EGF receptor protein, may be functionally related to the mitogenic action of TNF in human fibroblasts

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF

The synthetic double-stranded RNA polyinosinate-polycytidylate [poly(I).poly(C)] was mitogenic in cultures of human foreskin fibroblasts, as demonstrated by a stimulation of 3H-thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)-beta in human fibroblasts. Single-stranded poly(l) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)-beta, added to poly(I).poly(C)-stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN-beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN-beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)-stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN-beta can exert negative feedback control of cell proliferation