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.     

Evidence for cytostatic T cell activity in the effector mechanism against syngeneic TMT mammary tumor cells in mice

The effector mechanism of immune spleen cells against syngeneic TMT mammary tumor cells was analyzed in vitro. C3H/He mice were first inoculated with TMT tumor cells, and then the tumors were x-irradiated with 2000 rad 1 wk after the inoculation. Spleen cells from these treated mice inhibited the growth of tumor cells in vitro when assessed by (3H)-TdR incorporation by tumor cells (cytostatic activity). The same spleen cells did not have any cytotoxic activity on TMT tumor cells detected by a 51Cr-release assay. The cytostatic activity was mediated by Lyt-1+23- T cells. The purified T cells alone could not inhibit the growth of tumor cells, but accessory cells were required for the induction of cytostatic T cell activity. The accessory cells were Ia-positive, macrophage-like adherent cells. Furthermore, both T cells and macrophages were also required for the inhibition of tumor growth even after the spleen cells were activated in vitro. These results suggest T cells and macrophages play an important role in the effector mechanism against TMT mammary tumor cells. The mechanism of cytostasis by T cells and macrophages was discussed from the standpoint of the cellular interaction.     

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.     

Anti-tumor cytostatic mechanism and delayed-type hypersensitivity against a syngeneic murine tumor. Comparison between neonatally thymectomized mice and congenitally athymic nude mice

We studied the anti-tumor mechanism against a syngeneic tumor using a BALB/c-MA tumor system by cytolysis and cytostasis assays in vitro comparing mice neonatally thymectomized at 1 day or 7 days after birth (NTx-1, NTx-7), sham-operated (sham) mice, and congenitally athymic nude BALB/c mice. NTx-1 mice showed more rapid tumor growth and a slightly lower degree of strong cytostatic activity in peritoneal exudate cells (PEC) than NTx-7 or sham mice. Nude mice showed more rapid MA growth than NTx-1 mice and no cytostatic activity in PEC. After immunization with mitomycin C-treated MA (MMC-MA), NTx-1 mice acquired an immunoprophylactic capacity against MA and showed cytostatic activity and delayed footpad reaction (DFR) to MA, however, nude mice showed no acquisition of such an immunity, or cytostatic activity, or DFR to MA. These differences between NTx-1 and nude mice could be well-explained by less capacity of nude mice to produce a macrophage-activating factor, which activates macrophages to exert cytostasis and DFR. However, NTx-1 mice could not reject MA by immunization with MMC-MA in CFA (MMC-MA/CFA), although such immunized sham mice could eliminate MA completely. Both PEC and spleen cells from Sham mice immunized with MMC-MA/CFA showed cytostatic activity, whereas NTx-1 mice showed cytostatic activity of the same level in PEC and less in spleen cells compared to Sham mice. Cytolytic activity was never detected throughout this study in a BALB/c-MA system. These data suggest that cytostasis plays an important role in antitumor immunity against a syngeneic MA tumor and that two types of cytostasis is included from the standpoint of thymus-dependency of ontogenic development, relatively low and high.     

Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin

Desferal is a clinically approved iron chelator used to treat iron overload. Doxorubicin is an anthracycline cancer chemotherapy drug used in the treatment of breast cancer. It can undergo redox cycling in the presence of iron to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the cardiotoxic side effects of the drug, but it is unclear whether it is also required for its anti-tumor activity. To test whether an iron-chelating antioxidant would interfere with the tumor-killing activity of doxorubicin, nude mice were transplanted with xenografts of human breast cancer MDA-MB 231 cells and then treated with doxorubicin and/or desferal. Not only did desferal not interfere with the anti-tumor activity of doxorubicin, it inhibited tumor growth on its own. In vitro studies confirmed that desferal inhibits breast tumor growth. However, it did not induce apoptosis, nor did it induce cell cycle arrest. Instead, desferal caused cytostasis, apparently through iron depletion. The cytostatic activity of desferal was partially ameliorated by pretreatment with iron-saturated transferrin, and transferrin receptor expression on breast cancer cells nearly doubled after exposure to desferal. In contrast to its effect on tumor cells, desferal did not inhibit growth of normal breast epithelial cells. The data indicate that the anti-tumor activity of doxorubicin is not dependent on iron-mediated ROS production. Furthermore, desferal may have utility as an adjunctive chemotherapy due to its ability to inhibit breast tumor growth and cardiotoxic side effects without compromising the tumor-killing activity of an anthracycline chemotherapy drug.     

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Corynebacterium parvum in the activation of peritoneal macrophages. II. Identification of distinguishable anti-tumor activities by macrophage subpopulations

Two different mechanisms of murine macrophage (MP) antitumor activity are described in this report. C. parvum-activated peritoneal MPs were tested for cytotoxic and cytostatic activity 4 days after ip immunization. Cytotoxic activity could be distinguished from cytostatic activity using two different assay protocols. When MPs were separated by 1g velocity sedimentation, cytotoxic MPs were confined to high velocity fractions. In contrast, cytostatic MPs were found in cell fractions with velocities as low as 5.2 mm/hr. These two MP activities were also distinguishable by culturing at 37 degrees C for 24 hr. Cytotoxicity was abrogated when MPs were incubated in MEM, or MEM supplemented with lymphokine (LK) or indomethacin. In contrast, cytostasis remained at high levels when the cells were incubated with LK or indomethacin. Cytotoxicity was not retained after overnight culture even if LPS was present, or if various spleen or non-adherent peritoneal exudate cells were cocultured with the cytotoxic effector cells. Assays done to determine the presence of suppressor cells failed to find any inhibitory cell type. The phagocytic index, acid phosphatase activity, and H2O2 secretion were also measured before and after overnight culture. Acid phosphatase and phagocytic activities did not decline whereas H2O2 secretion declined significantly. These data indicate that in response to C. parvum, at least two different effector cell types with distinct antitumor activities are generated. Cytotoxicity, like the ability of cells to secrete H2O2, is found to be a short-lived function of CP stimulated MPs. In contrast, cytostasis is a function retained longer by MPs in culture.     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.     

Synergistic interactions of interleukin 1, interferon-beta, and tumor necrosis factor in terminally differentiating a mouse myeloid leukemic cell line (M1). Evidence that interferon-beta is an autocrine differentiating factor

The effect was investigated of combinations of cytokines known to be cytostatic for some tumor cells, namely interleukin 1 alpha (IL-1 alpha), interferon-beta (IFN-beta), and tumor necrosis factor (TNF), on the growth and differentiation of the mouse myeloid leukemic cell line, M1, cells. IL-1 alpha, IFN-beta, and TNF by themselves are antiproliferative for M1 cells. Treatment of cells with a mixture of any two of the three cytokines resulted in at least additive growth inhibition. None of these cytokines by themselves induced differentiation of M1 cells as assessed by increased expression of Fc receptors (FcR), stimulation of phagocytic activity and by morphologic criteria. However, as little as 1 U/ml IL-1 alpha in conjunction with IFN-beta or TNF increased FcR expression, phagocytic activity and morphologic changes in addition to inhibiting the growth of M1 cells. The combination of IFN-beta and TNF did not induce differentiation, although the growth of the cells was markedly inhibited. Both TNF and lipopolysaccharide (LPS) induced the in vitro production of IFN activity by M1 cells. Furthermore, the induction of differentiation of M1 cells by a combination of IL-1 alpha with either IFN-beta, TNF, or LPS was inhibited by antibody against mouse IFN-beta. Therefore, it appears that IFN-beta provides one of the two required signals for differentiation of M1 cells by these combinations of stimulants, the other being IL-1. Furthermore, the cytostatic effect of TNF by itself on M1 cells was also partly blocked by anti-IFN-beta antibody, suggesting that IFN-beta is also involved in the growth inhibitory effect of TNF for M1 cells. In contrast, the cytostatic effect of IL-1 on M1 cells was not blocked by anti-IFN-beta antibody. In conclusion, both the cytostatic and differentiative effect of TNF appear to be mediated by IFN-beta. Thus, the combination of IL-1 and IFN-beta or inducers of IFN-beta resulted in terminal differentiation of M1 cells. Northern blot analysis using cDNAs for murine IFN-beta1 or human IFN-beta2 showed an increased expression of mRNA for IFN-beta1 but not for IFN-beta2 by stimulation with TNF or LPS, strongly suggesting that IFN-beta 1 rather than IFN-beta 2 is responsible for TNF or LPS effects.     

Cytostasis of tumor cell lines by human granulocytes

Purified peripheral blood granulocytes from normal adult donors were tested for cytolytic and cytostatic activity against a variety of tumor-derived, virus-transformed, and normal cell lines. Altogether, 45 donors and 16 cell lines were tested. Although granulocytes mediated antibody-dependent cell-mediated cytolysis, no spontaneous cytolysis, as measured by chromium-51 (⁵¹Cr) or [³H]thymidine ([³H]TdR) release could be detected in assays performed for up to 12 hr, even at an effector:target (E:T) cell ratio of 100:1. In contrast, granulocytes exhibited substantial growth-inhibitory activity (GIA) against most target cells, as measured by uptake of [³H]TdR by the target cells. These results were confirmed by visual counting of target cells. The degree of cytostasis was dependent on the E:T ratio, with a plateau of 80–95% inhibition usually reached at a ratio of 40:1. Inhibition of growth of adherent tumor target cells was accompanied by cell detachment, with both effects apparent by 5 hr and reaching a peak after 15 hr of incubation. With nonadherent targets, the onset and the peak of cytostasis were delayed, being observed after 8 and 24 hr, respectively. Growth of target cells remained inhibited for up to 4 days of culture. A wide variety of target cells were sensitive to granulocyte-mediated cytostasis, including tumor-derived human and mouse cell lines, lymphoblastoid cell lines from normal donors, and embryo fibroblasts. Normal human fibroblasts were inhibited only at high E:T ratios (40:1). PHA-induced lymphoblasts were the only target cells tested that were completely resistant to the cytostatic effects of granulocytes and in fact, their growth was slightly stimulated. There appeared to be two somewhat different mechanisms of growth inhibition by granulocytes, which varied with the target cell. Trypsinization of granulocytes markedly reduced their reactivity against adherent target cells but had little effect on GIA against suspension target cells. Also, the activity against F-265, but not against other target cells, was almost completely abrogated in the presence of catalase, suggesting an important role of hydrogen peroxide in one mechanism of granulocytemediated cytostasis.     

Macrophage cytokines render WEHI-3B tumor cells susceptible to cytostasis by prostaglandins

The growth of the murine myelomonocytic leukemia tumor, WEHI-3B, has been shown to be inhibited by a two-step treatment: first, incubation for one hour with either interleukin-1 (human recombinant IL-1 alpha or tumor necrosis factor (human recombinant TNF-alpha); second, subsequent exposure to prostaglandins. Preincubation with IL-1 rendered the tumor cells more susceptible to subsequent treatment with either prostaglandin E2 or to the stable synthetic analogue of prostacyclin DC-PGI2. Preincubation with TNF-alpha rendered the tumor cells more susceptible to further treatment with PGE2 but not with DC-PGI2. Preconditioning of the tumour cells with either IL-1 alpha or TNF alpha did not affect cytostasis by subsequent culture of tumor cells in presence of either one of the cytokines. It is concluded that the interactions between macrophage cytokines and prostaglandins in enhancement of antitumor activity might imply first binding or induction of certain modifications in the tumor cells by the cytokines which render the cells more susceptible to exposure to prostaglandins.     

Tumor cell cytostasis by macrophages and antibody in vitro. II. Isolation and characterization of suppressed cells

Tumor cells in a cytostatic state caused by macrophages and antibody were isolated. Such suppressed cells excluded vital dye, incorporated uridine and leucine, and metabolized glucose. They did not, however incorporate thymidine, nor did they resume cell division in culture. During prolonged culture, these cells eventually died. In this system, cytostasis was an all-or-nothing phenomenon at the level of the individual cell. Once in the cytostatic state tumor cells did not resume proliferation.     

Direct activation in vitro of mouse peritoneal macrophages by pyran copolymer (NSC 46015)

Normal resting macrophages were transformed to cytostatic effector cells in the presence of pyran copolymer (NSC 46015) in the culture medium. Macrophage “activation” to inhibit MBL-2 leukemia cell growth was sharply dose-dependent and required >24 hr after exposure to pyran. The observed growth inhibition appeared to result from a modification of the macrophages themselves, since neither allogeneic macrophages nor drug alone interfered with MBL-2 cell growth. The primary mechanism of cytostasis did not involve phagocytosis or soluble macrophage product(s). Similar activation was observed for poly(I)·poly(C) and to a lesser extent for dextran sulfate. It is suggested that the antitumor activity of these polyanions is due to their function as direct macrophage stimulants.     

Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c-myc messenger RNA

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK-MEL-109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK-MEL-109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose-dependent way. Treatment with 10 ng/ml of TNF resulted in a two-fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c-myc mRNA was increased five-fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c-myc mRNA.     

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition

Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor.     

Induction of cytotoxicity of peritoneal exudate cells by agrimoniin,a novel immunomodulatory tannin of Agrimonia pilosa Ledeb

Summary The cytotoxic activities of the PEC after an i.p. injection of agrimoniin, a tannin contained in Agrimonia pilosa Ledeb. were studied. The plastic nonadherent PEC had significantly higher NK cell activity than the untreated control, and the adherent PEC were cytostatic toward MM2 and MH134 cells. The adherent PEC did not cause tumor cell lysis by themselves, but were cytolytic against MM2 cells in the presence of anti-MM2 sera. In the course of these effects of PEC after the i.p. injection of agrimoniin, the augmentation of NK cell activity was the earliest reaction, reaching a peak at 2 days after the injection; then, cytostatic activity increased. The induction of antibody-dependent cell lytic activity was a later reaction, which reached a peak at 6 days after the injection. Abbreviations used: PEC, peritoneal exudate cells; NK cell, natural killer cell; ADCC, antibody-dependent cell-mediated cytotoxicity; PMN, polymorphonuclear leukocytes     

Growth-stimulating, cytostatic and cytotoxic activity of the secretory products of the RPMI-6410t human B-cell lymphoblastoid line

The cells of human lymphoblastoid line RPMI-6410t were shown to synthesize constitutively a factor(s) with different types of biological activity. The factor(s) stimulated the growth of both B cells 6410t, obtained from the blood of a patient with acute myeloblastic leukemia, and the human embryonic diploid fibroblasts. With B cell lines Raji and P3HR-1.G5, obtained from the patients with Burkitt's lymphoma. The growth factor(s) displayed cytotoxic and cytostatic effects, respectively. Growth-stimulating and cytotoxic activating of the factor were destroyed by a 15 hour exposure to low or high pH. The activity was stable within pH values of 6-8. With regard to heat stability, the activity destroyed at 70 degrees C within 1 hour but remained stable at 56 degrees C during 1 hour. The above factor(s) displayed biological activities similar to those of the previously known tumor necrosis factor (TNF).