Potentiation of lymphokine-induced macrophage activation by tumor necrosis factor-alpha

In this study, we examined the possible role of TNF-alpha and lymphotoxin (TNF-beta) as cofactors of macrophage activation. The results demonstrate that both TNF were capable of enhancing the cytostatic and cytolytic activity of murine peritoneal macrophages against Eb lymphoma cells. The potentiation of tumor cytotoxicity became apparent when macrophages from DBA/2 mice were suboptimally activated by either a T cell clone-derived macrophage-activating factor or by IFN-gamma plus LPS. Neither TNF-alpha nor TNF-beta could induce tumor cytotoxicity in IFN-gamma-primed macrophages, indicating that TNF cannot replace LPS as a triggering signal of activation. In LPS-resistant C3H/HeJ macrophages, which were unresponsive to IFN-gamma plus LPS, a supplementation with TNF fully restored activation to tumor cytotoxicity. Furthermore, TNF-alpha potentiated a variety of other functions in low-level activated macrophages such as a lactate production and release of cytotoxic factors. At the same time, TNF-alpha produced a further down-regulation of pinocytosis, tumor cell binding and RNA synthesis observed in activated macrophages. These data demonstrate new activities for both TNF-alpha and TNF-beta as helper factors that facilitate macrophage activation. In particular, the macrophage product TNF-alpha may serve as an autocrine signal to potentiate those macrophage functions that were insufficiently activated by lymphokines.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

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.     

Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Evidence for the existence of two forms of membrane tumor necrosis factor: an integral protein and a molecule attached to its receptor

Plasma membranes were isolated from thioglycolate-induced peritoneal mouse macrophages and tested directly in a 51Cr-release assay against WEHI 164 tumor cells. These membranes showed anti-TNF antibody inhibitable killing of the TNF-sensitive tumor cell line, indicating that membrane-associated TNF is present on mouse macrophages. In order to elucidate whether membrane TNF is an integral protein or a molecule attached to a receptor, cells and plasma membranes were treated with low pH buffer. A partial reduction in TNF activity was observed which could be restored by incubation with exogenous TNF. In a Western blot analysis the integral membrane TNF could be identified as the 26-kDa molecule on activated mouse macrophages. These results indicate that both forms of membrane-associated TNF exist on macrophages and are responsible for cell-mediated cytotoxicity against TNF-alpha-sensitive targets.     

Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha.

Pretreatment with recombinant human granulocyte CSF (G-CSF) protected mice in two different models of septic shock. Intravenous injection of 250 micrograms/kg G-CSF to mice prevented lethality induced by 5 mg/kg LPS. Injection of 50 micrograms/kg G-CSF protected galactosamine-sensitized mice against LPS-induced hepatitis. In either case, this protection was accompanied by a suppression of LPS-induced serum TNF activity. In contrast, when galactosamine-sensitized mice were pretreated with 50 micrograms/kg murine recombinant granulocyte/macrophage CSF instead of G-CSF and subsequently challenged with LPS, serum TNF activity was significantly enhanced and mortality was increased. The suppressive effect of G-CSF on LPS-induced TNF production was also demonstrated in rats. In vivo, no TNF was detectable in the blood of LPS-treated rats, which had been pretreated with G-CSF. Ex vivo, alveolar macrophages, bone marrow macrophages, Kupffer cells, or peritoneal macrophages prepared from G-CSF-treated rats produced significantly less TNF upon stimulation with LPS than corresponding populations from control rats. However, when these macrophage populations were incubated with G-CSF in vitro, LPS-induced TNF production was unaffected. These data suggest that the G-CSF-mediated suppression of TNF production is not a direct effect of G-CSF on macrophages. To examine whether, independent of the protection against LPS, G-CSF treatment still activated neutrophils, it was demonstrated that granulocytes from G-CSF-treated rats were primed for PMA-induced oxidative burst and for ionophore/arachidonic acid-stimulated lipoxygenase product formation. The experiments of this study support the notion that G-CSF is a negative feedback signal for macrophage-derived TNF-alpha production during Gram-negative sepsis.     

Differences in the tumor necrosis factor-alpha-mediated lysis by fixed natural cytotoxic cells and fixed cytotoxic macrophages

TNF-alpha has been shown to be associated with macrophage cell membranes in such a way as to retain cytolytic activity despite fixation of the macrophage effector cells with paraformaldehyde. In this paper we report that, similar to cytotoxic macrophages, natural cytotoxic (NC) cells also use cell-associated TNF to lyse sensitive target cells. However, in contrast to fixed cytotoxic macrophages, NC cells do not retain cytolytic activity after fixation with paraformaldehyde. Additionally, the cytolytic activity of paraformaldehyde-fixed NC cells is not increased by incubation with LPS or by incubation with rTNF before fixation. Western blot analysis indicates that, unlike macrophages, NC cells use a smaller (17 kDa) constitutively active form of TNF. These results indicate that, although both macrophages and NC cells use effector cell-associated TNF to mediate lysis of sensitive targets, the way in which TNF is associated with these two types of effector cells must be different.     

Characteristics of macrophage activation by gamma interferon for tumor cytotoxicity in peritoneal macrophages and macrophage cell line J774.1

We investigated the characteristics of macrophage-mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN-gamma). In PMP, MTC was demonstrated when they were stimulated with IFN-gamma for 12 hr (short-term stimulation) and was abrogated when they were stimulated for 48 hr (long-term stimulation). Enhanced H2O2 generation was observed in PMP activated by long-term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non-treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short- and long-term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN-gamma stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN-gamma for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN-gamma. The results suggest that TNF-like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.     

The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.

Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.     

Cytolysis of tumor necrosis factor (TNF)-resistant tumor targets. Differential cytotoxicity of monocytes activated by the interferons, IL-2, and TNF

Herein we demonstrate that IFN-alpha, IFN-gamma, and IL-2 can induce human peripheral blood monocyte-mediated lysis of tumor cells that are resistant to both the direct effects of TNF and to monocytes activated by TNF. Monocytes activated by TNF kill only TNF-sensitive tumor targets, whereas those activated by IFN and IL-2 can lyse both TNF-sensitive and TNF-resistant tumor targets. Monocyte cytotoxicity against TNF-sensitive lines induced by the IFN, IL-2, or TNF can be completely abrogated by the addition of anti-TNF antibodies. In contrast, anti-TNF antibodies have no effect on IFN- or IL-2-induced monocyte cytotoxicity against TNF resistant targets, confirming non-TNF-mediated lysis induced by lymphokine-activated monocytes. Neither induction of TNF receptors by IFN-gamma nor inhibition of RNA synthesis by actinomycin D increased the susceptibility of TNF-resistant tumor targets to TNF-mediated monocyte cytotoxicity. Thus, non-TNF-mediated modes of monocyte cytotoxicity are induced by IFN and IL-2, but not by TNF, indicating that different cytotoxic mechanisms are responsible for the lysis of TNF-sensitive and TNF-resistant tumor cells. In addition, these findings also suggest that TNF-sensitive lines are susceptible only to TNF-mediated killing and apparently insensitive to non-TNF-mediated monocyte cytotoxicity.     

Hemorrhage induces enhanced Kupffer cell cytotoxicity while decreasing peritoneal or splenic macrophage capacity. Involvement of cell-associated tumor necrosis factor and reactive nitrogen.

Studies indicate that simple hemorrhage produces a profound depression of cell-mediated immunity, thereby contributing to an enhanced susceptibility to septic challenge in the host. However, it remains unknown whether or not the macrophages' cytotoxic capacity is altered after hemorrhage. To study this, C3H/HeN mice were bled to and maintained at a blood pressure of 35 mm Hg for 60 min, and adequately resuscitated. Mice were then killed at 2 or 24 h after hemorrhage to obtain peritoneal macrophage, splenic macrophage, and Kupffer cells. Cytotoxicity was assessed by determining the capacity of these macrophages to lyse [3H]TdR labeled WEHI-164 clone 13 or P815 tumor target cells (WEHI-164, sensitive to both soluble and cell-associated TNF vs P815 cells, insensitive to soluble TNF). Peritoneal and splenic macrophages from hemorrhaged animals exhibited a significantly reduced cytotoxic capacity, whereas Kupffer cells' ability to kill the target cells was enhanced. Similarly, the Kupffer cells' capacity to release TNF and IL-1, as well as express cell-associated forms of this cytokine are significantly enhanced on macrophages isolated 2 h after hemorrhage, whereas peritoneal macrophages are not. Furthermore, antibodies directed at mouse TNF but not against murine IL-1 alpha or murine IL-6 were able to oblate the enhanced target cell lysis of unfixed, as well as paraformaldehyde fixed (metabolically inactive) Kupffer cells. Studies using inhibitors (GN-monomethyl-arginine, superoxide dismutase, catalase, and ibuprofen) of other TNF-inducible mechanisms of target cell killing indicated that only the inhibition of the release of reactive nitrogen consistently depressed the cytotoxic capacity of Kupffer cells from hemorrhaged mice. Thus, the increased Kupffer cell cytotoxicity from hemorrhaged mice is most likely mediated through the expression of cell-associated TNF and the release of reactive nitrogen.     

Macrophage activation and immunomodulation by myeloperoxidase.

Myeloperoxidase (MyPo) is an enzyme found in neutrophils and monocytes that plays an important role in the microbicidal and cytocidal activities of these cells. The present studies show that this enzyme can also affect both capacities and functions of macrophages. When resident peritoneal macrophages from C57BL/6 mice were exposed to preparations of either human or canine enzyme in vitro, tumor necrosis factor (TNF) was released. The amount of TNF produced was dose dependent and could be neutralized with polyclonal anti-TNF. Low levels of interferon were also produced by these cells. In addition, exposure of murine macrophages in vitro to this enzyme resulted in increased ability to destroy 3T12 target cells. Intravenous injection of mice with myeloperoxidase induced the production of both TNF and interferon, which could be detected in the sera. Possible mechanisms of TNF induction include radical production by myeloperoxidase or ligand-receptor interaction by the binding of this enzyme to the mannosyl-fucosyl receptor. These results, when taken in their entirety, suggest that this enzyme can modulate the immune response through effects on macrophage function.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

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.     

Characteristics and mechanism of neutrophil-mediated cytostasis induced by tumor necrosis factor

After being treated with rTNF, polymorphonuclear neutrophils (PMN) were highly suppressive to the growth of four different tumor target cells, Raji, K562, UCLA-SO-M14, and U937. Neutralizing TNF with specific antibodies before PMN were treated blocked induction of the anti-proliferative activity against Raji. However, after PMN were exposed to TNF the cytostatic activity could not be reversed by the antibody or by washing off TNF, indicating that the continuous presence of TNF was not required for expression of the anti-proliferative function. Addition of the hydrogen peroxide (HP) scavenger, catalase, at the beginning of the assay inhibited the cytostatic activity, suggesting that HP was involved in suppressing the tumor cell growth. In contrast, other reactive oxygen species inhibitors such as superoxide dismutase, sodium azide, L-methionine, or deferoxamine did not inhibit the cytostasis. HP alone at above 10 microM was cytostatic to Raji cells. The presence of TNF did not increase the sensitivity of Raji to HP. TNF activated PMN to produce HP but the amount of HP released in the culture supernatant was too low for direct cytostasis. PMN also became more adherent after TNF treatment. Therefore, the TNF-induced cytostasis may be mediated by local high concentrations of HP produced by PMN.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.     

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF.

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-gamma in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF)-fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS +/- IFN-gamma in the presence of cycloheximide or activated just with IFN-gamma their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were "down-regulated" by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.     

A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.