Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.     

Natural cytotoxic activity in a cloned natural killer cell line is mediated by tumor necrosis factor

The interleukin-2-dependent mouse natural killer (NK) cell line NKB61A2 concomitantly exhibits NK and natural cytotoxic (NC) activities. This was determined by the cells' ability to lyse both the NK-sensitive YAC-1 lymphoma and the NC-sensitive WEHI-164 fibrosarcoma cell lines in a 4- and 18-hour 51Cr release assay, respectively. Cell-free supernatant from NKB61A2 cells grown in culture for 48 h had substantial lytic activity against WEHI-164. The mouse mast cell line PT18-A17 and the rat basophilic leukemia cell line RBL-2H3, which both express NC activity, also produced a soluble factor during culture which lysed WEHI-164 cells. This activity was increased in the basophilic/mast cells by crossbridging the surface IgE receptors. Similar results were obtained by triggering the basophilic NC cells with the calcium ionophore ionomycin and the tumor promoter phorbol-12-myristate-13-acetate (PMA). Such triggering of NKB61A2 cells, however, did not significantly increase their NC activity. Interestingly, both ionomycin and PMA had an inhibitory effect on the NK activity of NKB61A2. Recently it has been found that tumor necrosis factor (TNF) is a major mediator of NC activity. To determine if the soluble factor responsible for the NC activity of the NK clone was related to TNF, a rabbit polyclonal antiserum to mouse TNF was tested against the cell-free culture medium of NKB61A2, PT18-A17, RBL-2H3 and murine recombinant TNF (Mu-rTNF). The lytic activity of the culture medium from all these cells and the Mu-rTNF control was abrogated by this antibody. These data suggest that the murine cell line NKB61A2 has both NK and NC activities and that the NC activity is due to a factor immunologically similar to TNF. In addition, the enhancement of NC activity in the NK cell line is apparently under control by a separate pathway, different from that in the basophilic cells.     

Dendritic cells but not macrophages are targets for immune regulation by natural killer cells

Both dendritic cells (DC) and macrophages (M phi) stimulate lymphocyte proliferation in secondary mixed-lymphocyte (ML) reactions, though DC are approximately fourfold more effective. Natural killer (NK) cells suppress secondary ML reactions when DC are used, but NK cells do not suppress when M phi are used in these reactions. The findings are consistent with the idea that DC, but not M phi, are potential targets in immune regulation mediated by NK cells.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Effects of stress on lysability of tumor targets by cytotoxic T cells and tumor necrosis factor

The effects of stress on four tumor cell lines are analyzed in view of the possibility that stress protects tumor cells against immune attack. We show that stress causes resistance to CTL and TNF in two cell lines. Induction of resistance is time dependent and reversible and not due to failure of killer cells to interact with stressed targets. It is shown that stress induces stress proteins concomitant with induction of resistance to killer cells and TNF. Moreover experiments are presented suggesting that resistance to either immune effector is due to independent mechanisms. The conclusion that stress can induce mechanisms in targets that interfere with the action of TNF as well as with target lysis following a lethal hit by CTL is discussed.     

Tumor targets stimulate IL-2 activated killer cells to produce interferon-gamma and tumor necrosis factor

Lymphokine-activated killer (LAK) cells are cytotoxic for a variety of autologous and allogeneic tumor cells as well as modified autologous cells. It is assumed that LAK cells lyse their targets solely by direct cell to cell contact, possibly involving the degranulation and exocytosis of pore-forming elements, similar to that observed with cytotoxic T lymphocytes and NK cells. Reported here are studies demonstrating that LAK cells release factor(s) that are cytotoxic for a human breast carcinoma cell line, MCF-7, when stimulated with tumor cells. The factor(s) are slow acting and maximum cytotoxicity is observed only in a 72-h cytotoxic assay. The ability of LAK cells to secrete cytotoxic factor(s) is dependent on both the ratio of LAK cells to stimulating tumor cells as well as the length of their coincubation. A number of similarly slow acting cytokines that are cytostatic and/or cytotoxic for tumor cells have been described. We tested the ability of specific polyclonal antibodies directed against TNF, IFN-alpha, IFN-beta, and IFN-gamma to neutralize the cytotoxic supernatant activity. Only antibodies specific for IFN-gamma and TNF were neutralizing. We measured the amounts of IFN-gamma and TNF in the cytotoxic supernatants and determined that increased amounts of IFN-gamma and TNF were released after LAK cell-tumor cell interactions compared to supernatants of LAK cells alone or tumor cell alone. Comparable concentrations of human rIFN-gamma and rTNF resulted in similar levels (50 to 90%) of MCF-7 cell cytotoxicity as those observed with the stimulated LAK cell supernatants. We thus concluded that the majority of the cytotoxic activity released by LAK cells when stimulated with tumor cells was attributed to the synergistic activities of IFN-gamma and TNF. The significance of these observations in relation to the possible mechanisms by which LAK cells mediate cytolysis is discussed.     

Peyer's patch lymphocytes express natural cytotoxicity but not natural killer activity

The distribution of natural cytotoxic (NC) cells in the gut-associated lymphoid tissues (GALT) and in peripheric lymphoid organs was analyzed in comparison to that of natural killer (NK) cells. It was found that cells from the intestinal epithelium, mesenteric lymph nodes and spleen possess significant levels of NC and NK activity, whereas in thymus and popliteal lymph nodes both the natural activities are negligible. As previously shown for splenocytes, the NC activity of GALT cells is detectable in the 16-hour assays and not in the 4-hour assays. Interestingly, Peyer patches lymphocytes (PPL) possess extremely high NC activity but no NK activity. The NC activity of PPL is still high in NK-deficient mouse strains such as A/J and SJL/J. To further support the observation that the effector PPL are truly NC cells, it was shown that, as previously reported for spleen NC activity, overnight incubation at 37 degrees C of the lymphocytes only marginally affected the cytotoxicity of PPL, which could in turn be augmented by interleukin-3 (IL-3) containing supernatants. On the contrary, IL-2 could not increase NC or NK activity by PPL whilst augmenting NK activity of splenocytes. Thus, for the first time a cell population is identified which expresses only NC activity and not NK and which can be positively regulated only by IL-3.     

Morphology and lytic mechanisms of interleukin 3-dependent natural cytotoxic cells: tumor necrosis factor as a possible mediator

Populations of interleukin 3 (IL 3)-dependent cells can be derived from mouse bone marrow that display natural cytotoxicity (NC) against Wehi-164 target cells but do not display natural killing against YAC-1 cells. These bone marrow-derived NC cells cultured up to 2 mo in IL 3 do not contain rearranged T cell receptor beta-chain genes. They appear to be mast-like cells by electron microscopy and contain heterogeneous type granules. The molecules that mediate NC appear to be contained in these granules and are preformed because protein synthesis inhibitors have no effect on the capacity of IL 3-dependent NC cells to lyse Wehi-164 target cells. In addition to the IL 3-dependent bone marrow-derived cells, the basophilic leukemia cells, RBL-1, but not P815 mastocytoma cells were found to mediate NC against Wehi-164 cells. Both bone marrow-derived NC and RBL-1 cells can lyse L929 cells in 18 hr, suggesting that the putative NC mediator may be related to lymphotoxin/tumor necrosis factor (TNF). Recombinant human TNF displayed identical properties as NC cells; both entities possessed the same target cell specificity and had similar kinetics of target cell killing. The use of polyclonal rabbit antimouse TNF antibody blocked the actions of NC cells. Thus we believe that the mediation of NC is through the actions of a TNF-like molecule.     

Dexamethasone inhibits the cytotoxic activity of tumor necrosis factor

Effect of dexamethasone (DEX) on the cytotoxic activity of tumor necrosis factor (TNF) was examined using murine fibroblast cell line (L929 cells). DEX protected cells from the cytotoxic action of TNF. Protection of cytotoxic action was apparent when cells were pre-treated with DEX for 12h and no protection was observed in the presence of cycloheximide. These results suggested that de novo synthesis of new proteins was required for DEX-mediated protection. Moreover, prolonged simultaneous treatment with TNF and DEX resulted in the enhancement of cell growth, suggesting that TNF acted as a growth factor when cells were protected from the cytotoxic action of TNF. These results suggested that the signal transduction system for fibroblast growth enhancing and cytotoxic action of TNF were different from each other and that the interaction between TNF and glucocorticoids may play a modulating role in some inflammatory processes in vivo.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.     

Functional interaction between the tumor necrosis factor and interleukin 2 during induction of lymphokine activated killer cytotoxic activity

TNF has a differential effect on the induction of LAK activity which is dependent on the IL-2 concentrations used for LGL stimulation. TNF was found to synergize with low concentrations of IL-2 which are normally ineffective in inducing LAK activity. When IL-2 was used at concentrations optimal for LAK generation, TNF did not further enhance the differentiation of LGL into LAK effectors. When anti-TNF was included in the 51Cr-release assay, no effect on the cytolytic activity was observed. We also demonstrate that IL-2 induced an increase of specific TNF binding to LGL.     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Depressed natural cytotoxicity but normal natural killer cytotoxic factor (NKCF) production by mononuclear cells derived from patients with hepatocellular carcinoma

Summary This study investigated the relation between the production of natural killer cytotoxic factors (NKCF) and the phenomenon of natural killing (NK) activity against target K562 cells. Two different models of defective NK cell activity were employed. In the first instance, cytotoxic activity of mononuclear cells (MN) derived from patients with hepatocellular carcinoma was compared to the ability of these cells to produce NKCF. Although direct cytotoxicity was considerably impaired in these patients, the ability of their MN to produce NKCF when stimulated with K562 cells was found to be normal. In the second model, MN treated with the lysosomotropic drug monensin showed considerably reduced direct cytotoxic activity, although they were capable of producing normal amounts of NKCF when activated by K562 cells. These results therefore indicate that there is no correlation between NK activity and corresponding NKCF release, and suggest that NKCF production and activity is independent of direct NK cytotoxic activity.     

Human natural killer cytotoxic factor (NKCF): role of IFN-alpha

The relationship between production of NKCF and IFN-alpha by human lymphocytes was studied. NKCF activity was generated in response to K562-inducer cells. The presence of NKCF in supernatants was always accompanied by antiviral activity, but in several experiments IFN was detected without concomitant NKCF. In no instance was NKCF activity detected in the absence of IFN. Cell lines which were good inducers of IFN-alpha were found to be good inducers of NKCF. NKCF activity of supernatants was completely adsorbed after incubation with MOLT-4 cells, whereas there was only minimal depletion of IFN-alpha activity. Most of the antiviral activity and all of the NKCF activity of preformed supernatants was neutralized by anti-IFN-alpha serum, whereas anti-IFN-gamma serum and pH2 inactivation had minimal effect on either activity. Addition of IFN-alpha to neutralized supernatants restored NKCF activity. These experiments support the hypothesis that IFN-alpha is involved in the modulation of NKCF-lytic activity. Both antiviral and NKCF activities were abrogated when anti-IFN-alpha serum was added to cultures of lymphocytes plus inducer cells (induction phase). The addition of purified IFN-alpha to such cultures was effective in allowing resumption of NKCF activity; however, addition of IFN-gamma to these cultures did not overcome this block. The addition of purified IFN-alpha directly to supernatants generated in the presence of anti-IFN-alpha serum could not restore their NKCF activity, thereby suggesting an additional requirement for IFN-alpha in the production of NKCF. The possible role of IFN-alpha in the generation of NKCF and expression of its lytic activity is discussed.     

Analysis of a cytostatic lymphokine produced by incubation of lymphocytes with tumor cells: relationship to leukoregulin and distinction from recombinant lymphotoxin, recombinant tumor necrosis factor, and natural killer cytotoxic factor

Supernatants from the coculture of peripheral blood lymphocytes and the NK-susceptible cell line K562 were highly growth inhibitory for a variety of tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by NK cells. Rather, the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin, a recently described lymphokine. The supernatants of tumor-lymphocyte cultures contained only low levels of IFN-alpha and IFN-gamma, and antibodies to interferons did not affect the observed growth inhibition. The pattern of target cell susceptibility to growth inhibition by this factor was also quite distinct from that seen with purified recombinant LT or TNF. Furthermore, monoclonal antibodies to these cytokines also had no effect on the cytostasis, arguing against a requirement for, or synergistic interaction with, low levels of these cytokines. Some of the targets susceptible to the factor were only growth inhibited but not lysed, thereby distinguishing it from NKCF. Furthermore, the cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which have been reported to block NKCF. Therefore, the results show that a cytostatic factor is released in tumor-lymphocyte incubation that is quite distinct from interferons, LT, and TNF but has characteristics that resemble those of leukoregulin.     

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.