Protection from tumor necrosis factor cytotoxicity by protease inhibitors

Tumor necrosis factor (TNF) is cytocidal for human and murine cells when protein synthesis is inhibited by cycloheximide, but some protease inhibitors completely protect these cells from TNF cytotoxicity. Inhibitors of chymotrypsin-like proteases are active at lower concentrations than inhibitors of trypsin-like proteases. Both irreversible inhibitors, such as alkylating compounds, and reversible inhibitors, such as substrates of proteases, protect cells from the cytocidal activity of TNF. This protection is most effective when the cells are pretreated with these inhibitors before addition of TNF. When the protease inhibitors are removed, the cells gradually lose resistance to TNF cytotoxicity. The inhibitors do not interfere with the functioning of TNF-receptor complexes, since SK-MEL-109 melanoma cells treated with a protease inhibitor synthesize a TNF-induced protein. These findings suggest that a protease in involved in the cytocidal action of TNF.     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

Human monocytes cultured in a specially prepared medium free of lipopolysaccharide (LPS) constitutively produced a small, though significant, amount of tumor necrosis factor (TNF). Upon addition of LPS, the amount produced remained constant until the LPS concentration reached 1-10 ng/ml, whereupon the production of TNF dramatically increased, eventually becoming 100-fold greater than when the LPS concentration was below 1 ng/ml. Priming the monocytes with recombinant interferon-gamma (rIFN-gamma) before LPS exposure resulted in a 2- to 10-fold increase in TNF production, the highest relative increase being obtained at lower LPS concentrations and in the absence of LPS. Monocyte-produced TNF appears to be the effector molecule in monocyte-mediated killing of some target cell types, since antiserum against recombinant TNF inhibited killing of both actinomycin D-treated and untreated WEHI 164 cells by human monocytes. However, it also appears that TNF may not in all cases be an effector molecule in monocyte-mediated killing, since cytolysis of K562 cells mediated by IFN-gamma/LPS-activated monocytes was not inhibited by antiserum against recombinant TNF. Antiserum which was raised against a monocyte-derived cytotoxic factor and which neutralized recombinant TNF did, however, inhibit monocyte-mediated cytolysis of K562 cells, suggesting that an extracellular factor, perhaps related to TNF, was also involved in monocyte-mediated killing of K562 cells. A TNF-like activity was associated with the monocyte surface membrane, since paraformaldehyde-fixed monocytes expressed cytotoxic activity which was neutralized by antiserum against recombinant TNF. Fixed monocytes activated with rIFN-gamma in addition to LPS before fixation were generally more cytotoxic than those exposed to LPS alone, and those exposed to LPS were much more cytotoxic than those not exposed to LPS. Thus it is possible that high local TNF concentrations may be generated near the target cell upon direct contact between effector and target cells, and that also monocyte-associated TNF may in this way be involved in monocyte-mediated cytotoxicity.     

Reduction in tumor necrosis factor binding and cytotoxicity by hydrogen peroxide

The regulatory effect of H2O2 on both the cytotoxic activity and the specific binding of TNF-alpha was studied by using TNF-alpha-sensitized murine L929 cells. When these cells were exposed simultaneously to TNF-alpha and H2O2 (100 to 500 microM), the cytotoxic activity of TNF-alpha was inhibited by up to 66.6%. This inhibition was also effective when the cells were pretreated by H2O2, but not when TNF-alpha alone was preexposed to H2O2. These data suggest that H2O2 altered the cell sensitivity to TNF-alpha, without modifying the activity of the TNF-alpha molecule. Maximum loss of cell sensitivity to TNF-alpha occurred after 30-min preexposure to 500 microM H2O2. Complete restoration of TNF-alpha sensitivity was obtained within 12 h after H2O2 removal. It required protein synthesis as demonstrated by the suppressive effect of actinomycin D. The inhibitory effect of H2O2 was suppressed by catalase, but was unaffected by the scavengers of hydroxyl radical and hypochlorous acid, suggesting that H2O2 but not one of its metabolites was responsible for this inhibition. H2O2 inhibitory effect did not implicate any change in prostaglandin production or in PKC activity. In contrast, H2O2 effect was associated with an about 50% loss of the density of cell membrane 125I-TNF-alpha receptors (2949 vs 5620 binding sites per cell), without change in their affinity (3.9 vs 3.4 nM). Moreover H2O2 did not affect the rate of degradation of TNF-alpha, and only slightly increased the degree of internalization of 125I-TNF-alpha receptors. These findings indicate that H2O2 can down-regulate the cellular response to TNF-alpha, possibly by reducing the TNF-alpha-binding capacity.     

Reduction in tumor necrosis factor receptor affinity and cytotoxicity by glucocorticoids

Micromolar concentrations of glucocorticoids rendered L-M cells (a murine tumorigenic fibroblast line) less sensitive to the cytotoxic activity of murine TNF. The potency of different steroids paralleled their known anti-inflammatory potency, and pretreatment was more effective than post treatment. Sex steroids and mineralocorticoids were ineffective. Dexamethasone also decreased the sensitivity of MCF-7 (a human mammary carcinoma line) to the cytotoxic activity of human recombinant TNF. Pretreatment of both cell lines reduced the affinity of specific cell surface receptors for the binding of their species 125I-TNF about 3-fold while retaining the same number of binding sites. The decrease in sensitivity was not due solely to the inhibition of early TNF-induced events (such as binding, internalization or signal transduction). Dexamethasone modestly enhanced inhibition beyond that of neutralizing antiserum alone when both were added midway in the L-M killing reaction (after receptor down regulation but before the onset of complete cell death).     

Regulation of lymphocyte tumor necrosis factor receptors by IL-2

Activated lymphocytes are known to express TNF receptors. The precise stimuli involved in induction and regulation of these receptors have not been elucidated. Our findings demonstrate that IL-2, alone and in serum-free conditions, can trigger and regulate TNF receptor expression on normal lymphocytes. Flow cytometric analyses demonstrated that the receptor was rapidly induced on CD4, CD8, and CD16+ cells after IL-2 stimulation. Receptors increased with culture duration, became maximal between days 5 and 9, and were maintained for 18 to 20 days in the presence of IL-2. By using 125I-TNF and FITC-TNF binding, we present evidence that IL-2 concentration determines the magnitude of lymphoid TNF receptor expression--influencing both the percentage of TNF-positive cells within the population and the number of receptors/cell. Collectively, our results are persuasive for consideration of IL-2 as a central mediator in the regulation of lymphocyte TNF receptors.     

Regulation of macrophage tumor necrosis factor production by prostaglandin E2

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.     

Peroxidases enhance macrophage-mediated cytotoxicity via induction of tumor necrosis factor

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

The p70 tumor necrosis factor receptor mediates cytotoxicity.

Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (p55) and 70-80 kd (p70), mediate TNF action. Mouse TA1 cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TA1 cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.     

Role of tumor necrosis factor in monocyte/macrophage tumor cytotoxicity in vitro

The ability of activated monocytes/macrophages to exert cytotoxic effects in vitro which are preferentially manifest on target cells displaying a transformed phenotype has elicited intense efforts aimed at a molecular characterization of the underlying mechanism. This multistep reaction is typified by an apparently stringent requirement for conjugation between the effector and target to facilitate cytotoxicity, which has therefore long caused bias against the role of soluble effector molecules in mediating target cell damage. However, several laboratories have recently demonstrated a compelling role for at least one such mediator, tumor necrosis factor (TNF), in cell-mediated cytotoxicity exerted against certain target cells; these studies indicated that specific anti-TNF antibodies could block direct monocyte/macrophage-mediated cytotoxicity of TNF-sensitive targets. More recently we have shown that some targets which are completely resistant to soluble or fluid-phase TNF are effectively lysed by a TNF-dependent mechanism upon coculture with activated macrophages under conditions in which conjugation is facilitated. Furthermore, macrophage-mediated cytolysis of both TNF-sensitive and TNF-resistant targets occurs independently of the action of secreted TNF via this mechanism. The purpose of this review is to consider the implications of distinct modes of effector cell delivery of TNF to the target for molecular characterization of the target injury phase of macrophage-mediated tumor cytotoxicity.     

Regulation and dysregulation of tumor necrosis factor receptor-1

TNF is an essential regulator of the immune system. Dysregulation of TNF plays a role in the pathology of many auto-immune diseases. TNF-blocking agents have proven successful in the treatment of such diseases. Development of novel, safer or more effective drugs requires a deeper understanding of the regulation of the pro-inflammatory activities of TNF and its receptors. The ubiquitously expressed TNFR1 is responsible for most TNF effects, while TNFR2 has a limited expression pattern and performs immune-regulatory functions. Despite extensive knowledge of TNFR1 signaling, the regulation of TNFR1 expression, its modifications, localization and processing are less clear and the data are scattered. Here we review the current knowledge of TNFR1 regulation and discuss the impact this has on the host.     

Induction of tumor necrosis factor and cytotoxicity by macrophages exposed to lactoperoxidase and microperoxidase

Peroxidases are involved in a number of cytotoxic reactions. Murine thioglycollate-induced peritoneal macrophages were exposed to either lactoperoxidase or microperoxidase in vitro. At a concentration of 17.7 microM, both of these enzymes induced between 100 and 200 units of tumor necrosis factor (TNF) per 0.1 ml. Furthermore, these substances were able to stimulate macrophages to kill 3T12 target cells in a dose dependent manner at concentrations of 0.177 microM to 17.7 microM. Per cent cytotoxicity varied from 20% up to 75% at the higher concentrations. It was concluded that these enzymes can induce macrophages to secrete TNF and become activated to the cytotoxic state.     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with recombinant tumor necrosis factor

The effect of murine rTNF-alpha on c-fos and TNF mRNA accumulation and protein synthesis was investigated in bone marrow-derived macrophages to examine the mechanism(s) by which TNF modulates macrophage activity. A rapid and transient expression of the c-fos gene was induced by murine rTNF. This was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, suggesting that the murine rTNF stimulated a protein kinase C-dependent signal transduction pathway. Although LPS induced the accumulation of one TNF mRNA species, murine rTNF induced the synthesis of two distinct TNF mRNA species. Both LPS- and murine rTNF-induced TNF mRNA accumulation was equally enhanced by pretreatment with mouse rIFN-gamma. In contrast, cycloheximide pretreatment had little effect on murine rTNF-induced TNF mRNA accumulation, whereas this treatment increased LPS-induced TNF mRNA by sevenfold. These results argue that TNF mRNA accumulation can be modulated in macrophages by distinct mechanisms. As assessed by Western blot and immunoprecipitation analysis, LPS stimulated the synthesis of both cell-associated and secreted forms of TNF protein. In comparison, newly synthesized TNF protein was not detected when macrophages were treated with murine rTNF alone or in combination with murine rIFN-gamma. This demonstrates that although murine rTNF stimulated the synthesis of two distinct TNF mRNA species, additional signal(s) are necessary for their translation into protein and that such signals are present after LPS stimulation.     

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.     

Regulation of death complexes formation in tumor necrosis factor receptor signaling

TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions. We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8. Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.     

Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity

To determine how hyaluronidase increases certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity, we report here the isolation and characterization of a hyaluronidase-induced murine WW domain-containing oxidoreductase (WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear localization sequence, and a C-terminal alcohol dehydrogenase (ADH) domain. WOX1 is mainly located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain. Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside resulted in WOX1 release from mitochondria and subsequent nuclear translocation. TNF-mediated WOX1 nuclear translocation occurred shortly after that of nuclear factor-kappaB nuclear translocation, whereas both were independent events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH domains as determined using stable cell transfectants. In parallel with this observation, WOX1 also enhanced TRADD (TNF receptor-associated death domain protein)-mediated cell death in transient expression experiments. Antisense expression of WOX1 raised TNF resistance in L929 cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-x(L) (>85%), but up-regulation of pro-apoptotic p53 ( approximately 200%) by the ADH domain. When overexpressed, the ADH domain mediated apoptosis, probably due to modulation of expression of these proteins. The WW domains failed to modulate the expression of these proteins, but sensitized COS-7 cells to TNF killing and mediated apoptosis in various cancer cells independently of caspases. Transient cotransfection of cells with both p53 and WOX1 induced apoptosis in a synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds to the proline-rich region of p53 via its WW domains. Blocking of WOX1 expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is a mitochondrial apoptogenic protein and an essential partner of p53 in cell death.     

Stimulation of neutrophils by tumor necrosis factor

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.