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.     

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.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.     

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.     

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.     

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

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.     

Destruction of tumor cells by monokines released from activated human blood monocytes: evidence for parallel and additive effects of IL-1 and TNF

Summary The incubation of human peripheral blood monocytes with endotoxins activates the cells to lyse tumorigenic targets directly and also induces the production and release into the culture medium of factors that produce lysis of mouse-transformed fibroblasts L-929 (tumor necrosis factor (TNF)-sensitive) and human A-375 melanoma cells (interleukin-1 (IL-1)- and TNF-sensitive). Immunoblotting analysis revealed that the culture medium of endotoxin-activated but not of control monocytes contained both IL-1 and TNF with a molecular weight of 17,000 daltons each. TNF activity was determined by lysis of L-929 cells, and IL-1 activity was measured by the proliferation of D-10 cells. The production of IL-1 and TNF was concentration-dependent, and the amounts of these monokines were paralleled. The antitumor activity of the culture supernates from endotoxin-treated monocytes was significantly decreased by incubation with heterologous antisera to IL-1, TNF, or both. Recombinant human IL-1 and TNF were used in parallel experiments and as positive controls. Each monokine used produced cytotoxic effects in susceptible targets. The combination of IL-1 and TNF, which more likely resembles culture supernates of activated macrophages, produced an additive antitumor cytotoxicity effect.     

Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.     

Enhancement of human monocyte tumoricidal activity by recombinant M-CSF

Activated monocytes are an important component of immunologic defense against neoplastic disease. A variety of agents capable of inducing tumoricidal activity have been described, including bacterial LPS, IFN-gamma, IL-1, IL-2, TNF, and GM-CSF. We now show that pretreatment of monocytes with recombinant human macrophage-specific colony stimulating factor (M-CSF) augments the tumoricidal activity of human peripheral blood monocytes induced by other activating agents. Monocytes were preincubated for three days with M-CSF at 10(3) U/ml, washed, and treated for an additional two days with secondary activators. Tumoricidal activity was measured in a 6-h 51Cr-release assay using NK-resistant WEHI 164 cells that had been treated with actinomycin D. Pretreatment of monocytes with M-CSF significantly increased tumoricidal activity induced by LPS, IFN gamma, LPS plus IFN gamma, and LPS plus PMA. Pretreatment with IL-1, IL-2, IL-3, IL-4, or GM-CSF was not as effective as M-CSF in increasing tumoricidal activity. Enhanced tumoricidal activity was directly correlated to the increased TNF production resulting from M-CSF pretreatment. TNF antiserum completely blocked tumoricidal activity, demonstrating that TNF was responsible for the M-CSF-mediated increase in tumor cell lysis. M-CSF pretreatment also enhanced non-TNF mediated tumoricidal activity by monocytes, as seen by increased killing of the TNF-resistant target P815. This study demonstrated that in addition to the role of M-CSF in the proliferation and differentiation of monocyte/macrophage precursors, M-CSF also augments an effector function of mature blood monocytes.     

Plasma membrane-associated tumor necrosis factor. A non-integral membrane protein possibly bound to its own receptor

Purified plasma membranes from LPS-activated human blood monocytes produced significant lysis and growth inhibition of the TNF-sensitive L929 murine fibroblast cell line. Unactivated human monocyte plasma membranes did not display either activity. Anti-TNF serum specifically inhibited the anti-tumor activity of activated monocyte membranes whereas anti-IL-1 serum or non-specific rabbit serum decreased neither the lysis nor growth inhibition of L929 cells. Membrane-associated TNF did not behave as an integral protein as it could be eluted from the plasma membranes by either high salt or low pH treatment. Plasma membranes cleared of membrane-associated TNF by high salt treatment were able to bind TNF, and this binding was specifically inhibited by preincubation of rTNF with specific anti-TNF serum. Western blot analysis of plasma membranes showed a membrane-associated TNF with a m. w. of approximately 17 kDa present only in the activated monocytes. When the plasma membranes were preincubated with the cross-linker agent dissuccinimidyl suberate, Western blot analysis revealed the presence of a TNF-binding protein with a Mr of approximately 102 kDa. These studies indicate that unlike IL-1, membrane-associated TNF is not an integral membrane protein and that TNF may be associated with the monocyte membrane by occupying the TNF R.     

Mechanisms of TNF resistance: identification of membrane phosphoproteins associated with a dominant resistant phenotype in lymphoid-myeloid somatic cell hybrids

Analysis of 53 somatic cell hybrids between TNF-sensitive myeloid cells (U937) and TNF-resistant T-cell lines HUT78 (UH-hybrids) and Jurkat (UJ-hybrids), respectively, revealed complete resistance to TNF-mediated cytostasis in all cases. Moreover, all hybrids remained insensitive to a combined treatment with TNF-alpha and IFN-gamma, which exert synergistic growth inhibition and cytotoxicity on parental U937 cells. Analyses of cell surface marker expression, membrane phosphoproteins, and expression of tissue-specific cytokine genes revealed differential conservation of myeloid and T-cell-specific properties in each of these hybrids, but invariant, dominant resistance to TNF-alpha-mediated growth inhibition. All TNF-resistant hybrids expressed a membrane phosphoprotein pattern, which closely resembled that of the respective parental T-cell lines. In particular, two membrane phosphoproteins of apparent molecular weight of 50,000 and 38,000 were common in the two parental T-cell lines and all UH- and UJ-hybrid clones, suggesting a possible role of these proteins in mediating TNF resistance.     

Multiple cytolytic mechanisms displayed by activated human peripheral blood T cell subsets.

It has been proposed that CTL-mediated cytotoxicity may involve multiple lytic mechanisms. We have examined both the antibody-redirected cytolytic potential and the direct cytotoxicity of purified human peripheral blood high buoyant density CD4+ and CD8+ T cells activated with IL-2 and anti-CD3 mAb. TNF-sensitive and TNF-resistant targets and various metabolic inhibitors were used to compare the antibody-redirected cytotoxicity of T cell subsets and discern the role of potential lytic mediators. In a 4-h assay against several different nitrophenyl-modified targets, the heteroconjugated antibody (anti-CD3-anti-nitrophenyl) redirected cytolytic potential of 72-h activated CD4+ T cells was inhibited by the continuous presence of actinomycin D, cycloheximide, and EGTA, but not mitomycin C, cyclosporin A, or cholera toxin (CT). Conversely, only CT and EGTA inhibited the antibody-redirected cytolytic potential of activated CD8+ T cells. Despite both CD4+ and CD8+ T cell subsets expressing granzymes, pore-forming protein, TNF-beta, and TNF-alpha, these T cell subsets displayed distinct pathways of antibody-redirected lysis against TNF-sensitive and TNF-resistant targets, even in the presence of anti-TNF antibodies. In addition, these same effector T cell subsets were also directly cytotoxic (in the absence of heteroconjugated antibody) against TNF-sensitive targets in an 18-h assay. Indeed, this direct cytotoxicity was completely abrogated by anti-TNF-alpha antibody and was sensitive to the metabolic inhibitors (cyclosporin A, CT, cycloheximide, and actinomycin D), all of which blocked CD4+/CD8+ T cell TNF-alpha production. Therefore, both CD4+ and CD8+ T cells were demonstrated to utilize antibody and lymphokine-mediated lytic mechanisms. CD4+ and CD8+ effector subsets were demonstrated to lyse the same TNF-sensitive target by these two different mechanisms. Although it cannot be excluded that the redirected lytic mechanisms of both CD4+ and CD8+ effectors share common elements, it is likely that other important events in this cytolytic process are fundamentally distinct between these subsets of T cells.     

Expression of two distinct cytolytic mechanisms among murine CD4 subsets

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.     

Cytotoxicity of activated monocytes on endothelial cells

Unstimulated human monocytes did not express appreciable levels of cytotoxicity on normal human umbilical vein endothelial cells (EC) in a 24-48 hr TdR release assay. On activation with IFN-gamma and LPS, monocytes had appreciable cytotoxicity on EC. Monocyte cytotoxicity on EC was not dependent on the presence of contaminating lymphoid cells. Recombinant TNF, IL-1, and IL-6 as well as monocyte supernatants did not exert a cytotoxic effect on EC. Moreover, anti-TNF, anti-IL-1, and anti-IL-6 antibodies, as well as scavengers of reactive oxygen intermediates, did not affect the cytotoxicity of activated monocytes on EC. Antibodies against the beta-chain (CD18) of leukocyte integrins inhibited the adhesion and cytotoxicity of activated monocytes on EC. Pretreatment of EC with IL-1 augmented the adhesion of monocytes on EC. Normal monocytes were not cytotoxic on IL-1-pretreated EC and IL-1 treatment did not increase the susceptibility of EC to activated monocytes. Thus adhesion is necessary but not sufficient for monocyte killing of EC. Anti-alpha L (LFA-1) antibodies markedly reduced monocyte cytotoxicity on EC, although anti-alpha X (p150) antibodies had only a modest effect. Anti-alpha M (Mac-1/CR3) antibodies were intermediate inhibitors of EC killing by activated monocytes. Thus, alpha L, beta 2 (LFA-1), and, to a lesser extent, alpha M, beta 2 (Mac-1/CR3) and alpha X, beta 2 (p 150, 95) integrins are the main adhesive structures involved in the cytotoxic interaction of activated monocytes with EC. Monocyte-mediated damage of EC could play a role as a mechanism of tissue injury under conditions of local or systemic activation of mononuclear phagocytes.     

Tumor necrosis factor-mediated cytotoxicity by tumor-associated macrophages

We have directly demonstrated that macrophages present within solid EMT6 mammary tumors (of BALB/c origin) produce TNF-alpha (TNF). These tumor-associated macrophages lysed WEHI-164, a TNF-sensitive cell line, very efficiently. This cytotoxicity was abrogated in the presence of anti-TNF antisera. In contrast, EMT6 cells, the tumor from which the macrophages were obtained, were not effectively lysed by the macrophages and were 100-fold less sensitive to lysis by recombinant mouse TNF. Thus, marked heterogeneity exists among tumors regarding sensitivity to TNF-mediated cytotoxicity. Similarly, macrophages which infiltrate into EMT6 multicellular spheroids implanted into the peritoneal cavity as well as free cells within the cavity exhibited TNF-mediated cytotoxicity of WEHI-164 cells, but failed to lyse EMT6 cells. The kinetics of lysis by these cells was similar to that of recombinant mouse TNF.     

Tumor growth in vivo selects for resistance to tumor necrosis factor

The relationship between in vivo tumor growth and resistance to TNF in WEHI-164 cells has been examined. When a highly TNF-sensitive clone of WEHI-164 was grown in vivo in syngeneic mice it became resistant to rTNF such that a 4 to 5 log higher concentration of TNF was required to produce tumor lysis in vitro. When compared with an in vitro selected TNF-resistant variant, the in vivo selected line was significantly more tumorigenic. The resistant phenotype of both the in vivo and in vitro selected variants was stable in culture and both selected lines were also resistant to lysis by syngeneic spleen cells with natural cytotoxic activity. The parental clone and the two variants were equally sensitive to lysis by allo-CTL and expressed similar levels of MHC class I Ag. Resistance to TNF in the two variants was not a function of de novo production of TNF measured as supernatant TNF activity or TNF mRNA expression. These studies are the first to demonstrate that in vivo tumor growth results in resistance to TNF and therefore may have direct relevance to the efficacy of TNF in the treatment of human neoplasms.     

Natural cytotoxic cell-specific cytotoxic factor produced by IL-3-dependent basophilic/mast cells. Relationship to TNF

The murine IL-3-dependent mast cell line, PT18-A17, and the rat basophilic leukemia cell line, RBL-2H3, were found to mediate natural cytotoxic (NC) activity via the release of a soluble factor which specifically lysed NC-sensitive WEHI-164 but not NK-sensitive YAC-1 tumor cells. The release of this NC cell-specific cytotoxic factor was enhanced by triggering of both types of cells via IgE receptor bridging. This factor had activity on TNF-sensitive but not TNF-resistant cell lines and could be neutralized by two independently produced polyclonal anti-mouse TNF antisera. It was not neutralized by antibodies against mouse IFN-alpha/beta or IFN-gamma. Moreover, it was not neutralized by a monoclonal or a polyclonal anti-human TNF, demonstrating that the rodent TNF differed antigenically from human TNF. These results indicate that the cytotoxic factor released from a murine IL-3-dependent mast cell line and from a rat basophilic leukemia cell line is immunologically and functionally related to murine TNF.     

TPA induction of EL4 resistance to macrophage-released TNF: role of ADP-ribosylation in tumoricidal activities of TNF and other factors

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.