Faculty Bibliography

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

A protein termed IFN-beta 2, originally described on the basis of antiviral activity and antigenic cross-reactivity with the classical IFN-beta, is now known to be identical with the independently isolated B cell stimulatory factor (BSF-2). Earlier it was suggested that IFN-beta 2 (i.e., BSF-2) mediates the antiviral action of TNF in human fibroblasts. We examined Escherichia coli-derived recombinant preparations of human IFN-beta and BSF-2 for antiviral activity and plasmacytoma growth factor (PCT-GF) activity. IFN-beta had antiviral activity but showed no PCT-GF activity. BSF-2 showed potent PCT-GF activity but lacked antiviral activity. Antiviral activity of IFN-beta was neutralized by polyclonal antibodies and mAb to IFN-beta, but not by antibody to rBSF-2. PCT-GF activity of BSF-2 was neutralized by antibody to rBSF-2, but not by antibodies neutralizing the antiviral action of IFN-beta. Five mAb and a polyclonal antibody to human IFN-beta failed to react with BSF-2 in a solid phase RIA and antibody to BSF-2 did not react with IFN-beta. PCT-GF activity in supernatants of human FS-4 fibroblasts stimulated with TNF, IL-1 or poly(I).poly(C) was neutralized by antibody to rBSF-2, but not by antibodies neutralizing the antiviral activity of IFN-beta. Finally, the antiviral activity of TNF in FS-4 cultures was neutralized by antibodies to IFN-beta but not by antibodies to BSF-2. Taken together, these results support the view that the antiviral action of TNF in human fibroblasts is mediated by IFN-beta, and not by BSF-2/IFN-beta 2 that apparently lacks significant antiviral activity

The survival and proliferation of the murine plasmacytoma cell line T1165 was previously shown to depend on a plasmacytoma growth factor (PCT-GF) produced by the murine P388D1 macrophage cell line. In the present study we examined several cytokines for their ability to stimulate the proliferation of T1165 cells. Recombinant human interleukin 6 (IL-6; also termed BSF-2 or interferon-beta 2) exhibited a potent growth stimulating effect on T1165 cells, with a maximal stimulation observed at 2 ng/ml or higher concentrations. Recombinant tumor necrosis factor (TNF) and recombinant interleukin 1 (IL-1) were found to produce a similar growth stimulation. Both murine and human TNF induced a maximal or near-maximal DNA synthesis in T1165 cells at 10 to 100 ng/ml after a 24-hr incubation. Phorbol myristate acetate (PMA) caused a weak stimulation of DNA synthesis in T1165 cells, and combined treatment with TNF and PMA resulted in an additive stimulation. The promotion of T1165 cell proliferation by TNF appeared to be the result of a direct action, as no autocrine growth factor could be detected in T1165 cultures after incubation with TNF. These results indicate that TNF and IL-1 can substitute for IL-6 as essential growth factors in a growth factor-dependent murine plasmacytoma line

Tumor necrosis factor (TNF) induced loss of TNF receptors in HeLa cells was studied using acid elution technique, which could distinguish surface occupancy and real loss of receptors. Exposure of HeLa cells to TNF resulted in a rapid reduction in the number of TNF receptors without affecting the apparent binding affinity. The binding of transferrin after treatment with unlabeled TNF was unaffected. The TNF-mediated decrease in receptor number on the cells was reversible. Following removal of TNF from growth medium, binding activity was restored within 3 h. Cycloheximide prevented the restoration of TNF receptors, suggesting that de novo synthesis of receptors was required to restore the binding activity

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) were shown previously to be mitogenic for human fibroblasts. Here we show that recombinant human TNF and recombinant human IL-1 alpha increase steady state levels of c-fos and c-myc proto-oncogene mRNAs in quiescent human FS-4 fibroblasts. Proto-oncogene mRNA levels were enhanced within 20 min of TNF or IL-1 addition, peaked at 30 min, and declined to undetectable levels (c-fos) or basal levels (c-myc) by 60 or 90 min. A similar rapid increase in c-fos and c-myc mRNA was seen in quiescent FS-4 cells exposed to cycloheximide. However, in the presence of cycloheximide, both proto-oncogene mRNA levels continued to rise for at least 90 min. The transient nature of the increase in c-myc mRNA levels appears to be a response characteristic for TNF and IL-1 because in quiescent FS-4 cells exposed to 10% fetal bovine serum, steady state levels of c-myc mRNA remained elevated for at least 4 h