Effect of antisera against recombinant tumor necrosis factor and the monocyte-derived cytotoxin(s) on monocyte-mediated killing of various tumor cells

Antisera raised against recombinant tumor necrosis factor (TNF) and against the monocyte-derived cytotoxic/cytostatic protein factor (CF), which is related to recombinant TNF, have been compared with respect to their ability to inhibit monocyte-mediated killing of various types of cells which differ in their sensitivity to recombinant TNF. During 6 hr of coculturing monocytes and target cells, the recombinant TNF antiserum inhibited killing of the extremely TNF-sensitive WEHI 164 clone 13 cells and actinomycin D-treated WEHI 164 cells from which the clone 13 cells were derived (parental WEHI 164 cells (P-WEHI 164 cells]. The CF antiserum also inhibited monocyte-mediated killing of these cells during 6 hr of coculturing with monocytes, but on a per volume basis it was less potent than the recombinant TNF antiserum, consistent with the fact that the CF antiserum also was much less potent in inhibiting the cytotoxic activity of recombinant TNF. However, during 72 hr of coculturing with monocytes and target cells, the CF antiserum inhibited monocyte-mediated killing of P-WEHI 164 cells more efficiently than the recombinant TNF antiserum. Moreover, during 72 hr of coculturing with monocytes, only the CF antiserum was able to significantly inhibit monocyte-mediated killing of the relatively recombinant TNF-resistant K562 cells. This suggests that a factor immunologically different from recombinant TNF, perhaps a form of natural TNF differing somewhat immunologically from recombinant TNF, was involved in the killing of K562 cells, and possibly in the killing of P-WEHI 164 cells, during 72 hr of coculturing with monocytes. Although this factor was present extracellularly, it appears that it may act as a monocyte-associated factor in monocyte-mediated killing of K562 cells, since exposure to recombinant interferon-gamma (rIFN-gamma) in the absence of Escherichia coli endotoxin (lipopolysaccharide, LPS) activated the monocytes to mediate killing of K562 cells more efficiently than exposure to LPS alone, despite the fact that only little cytotoxic/cytostatic activity was released from the monocytes without the addition of LPS. The ability of rIFN-gamma and LPS to activate monocytes to produce and release CF has also been studied.     

Monocyte-mediated drug-dependent cellular cytotoxicity: effects on different WEHI 164 Target cell lines

Summary The contribution of monocyte cytotoxic protein factor (CF) to monocyte-mediated drug-dependent cellular cytotoxicity (DDCC) has been investigated. Cell lines which have been derived from murine WEHI 164 cells (termed WEHI 164 parental) by selecting for high (WEHI 164 clone 3) and low (R-WEHI 164) sensitivity to CF-mediated cytotoxicity were used as target cells in DDCC. By comparing the CF doses which produced 50% dead cells (LD 50) we found that WEHI 164 clone 3 was approximately 30 times more sensitive than WEHI 164 parental which in turn was 70 times more sensitive than R-WEHI 164. Actinomycin D (Act D) treatment of WEHI 164 parental and R-WEHI 164 greatly increase susceptibility to CF-mediated cytotoxicity. The susceptibility of WEHI 164 clone 3 was apparently somewhat increased at low dilutions of CF, whereas no significant increase was observed at high dilutions. The susceptibility to DDCC of the three target cell lines (WEHI 164 parental, WEHI 164 clone 3, and R-WEHI 164) correlated with the sensitivity pattern obtained in CF-mediated cytotoxicity of Act D-treated target cells. Monocyte- and CF-mediated cytotoxicity against Act D-treated WEHI 164 clone 3 and R-WEHI 164 was inhibited by neutralizing CF antiserum. These data indicate that CF is an effector molecule in monocyte-mediated DDCC.     

WEHI 164 sarcoma cells rendered resistant to monocyte-released cytotoxin are also resistant to monocyte-induced cytolysis

Summary WEHI 164 sarcoma cells cultured with monocyte-released cytotoxin (CF) for 4 weeks became resistant to CF-induced cytolysis and were concomitantly rendered resistant to monocyte-induced cytolysis. The resistant cell line (R-WEHI 164) has been stable with respect to resistance to monocyte- and CF-induced lysis for more than 7 months. WEHI 164 and R-WEHI 164 cells adsorbed CF and no significant difference in CF adsorption was observed. The results indicate that CF may be an effector molecule in monocyte-mediated cytolysis.     

Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cytotoxicity is independent of secretion of reactive oxygen intermediates and is suppressed by protease inhibitors

Pretreatment with Actinomycin D (ActD, 1 microgram/ml for 3 hr) rendered WEHI 164 tumor cells susceptible to killing by human monocytes in a 6-hr 51Cr release assay. The present study was designed to elucidate the role of reactive oxygen intermediates (ROI) and of proteolytic enzymes in this reactivity. ActD-treated WEHI 164 cells did not trigger any measurable release of O-2 or H2O2 from monocytes. Monocytes exposed to phorbol-12-myristate-13-acetate, which enhanced release of ROI, did not show augmented killing of ActD-treated tumor cells. Scavengers of oxygen metabolites (catalase, superoxide dismutase, gluthatione, and mannitol), which inhibited ROI-mediated PMA-induced monocyte cytotoxicity against erythrocytes, did not affect monocyte killing of ActD-treated WEHI 164 cells. Enzymatically generated ROI with xanthine/xanthine-oxidase glucose/glucose-oxidase did not show preferential killing of ActD-treated WEHI 164 cells. Two patients with chronic granulomatous disease had normal levels of monocyte cytotoxicity against ActD-treated tumor cells. To determine the possible role of proteolytic enzymes in mediating this reactivity, we studied various antiproteases. Organophosphorous agents (DFP and PMSF), chloromethyl-ketone derivatives of tosylamino acids (TLCK and TPCK), Actinomyces products (pepstatin and chymostatin), and the synthetic protease substrate TAME inhibited monocyte-mediated cytotoxicity against ActD-treated WEHI 164 cells. The macromolecular protease inhibitors alpha-1 antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor, and the synthetic protease substrate ATEE had little effect on monocyte cytotoxicity. When monocytes were preincubated with drugs for 1 hr and washed, TLCK, TPCK, and PMSF inhibited cytolysis, whereas the less effective chymostatin and TAME and the inactive BPTI had no effect under these conditions. Inhibition by preincubation with TLCK, PMSF, and TPCK was completely reversed after 6 hr of culture. Supernatants of monocyte cultures had lytic activity against ActD-treated WEHI 164 but not against untreated cells. Antiproteases inhibited the lytic activity of monocyte supernatants. These results strongly suggest that ROI do not play a critical role in monocyte-mediated rapid killing of drug-treated tumor cells, and that proteolytic enzymes are involved in this reactivity.     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

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

Cytotoxic effector function of B lymphoblasts

EBV-transformed B lymphoblastoid cell lines and clones were cytotoxic in vitro against Actinomycin D-pretreated WEHI 164 sarcoma cells, a system in which mononuclear phagocytes were previously identified as effectors. Similarly, unlike resting B cells, normal B lymphoblasts stimulated for 72 hr with Staphylococcus aureus Cowan I and purified according to the expression of the B cell surface marker B1, demonstrated appreciable cytolytic activity. B lymphoblastoid cells and derived supernatants were cytotoxic for Actinomycin D-pretreated WEHI 164 cells, and killing was inhibited by an anti-lymphotoxin but not an anti-tumor necrosis factor antiserum. Thus, B lymphoblasts have cytotoxic potential, mediated by lymphotoxin or a lymphotoxin-like soluble product.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

Human monocyte-mediated tumor cytotoxicity. I. Demonstration of an oxygen-dependent myeloperoxidase-independent mechanism

The cytolytic capacity of monocytes per se and stimulated monocytes has been documented to only a limited extent, and when observed has been ascribed to the generation of a variety of cytolytic molecular entities. In the present study we have examined de novo human monocyte-mediated tumor cytotoxicity and that induced by the agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Cytolytic function was analyzed by reference to the release of [111In] oxine from two prelabeled tumor cell lines, K562 and U937, in a 16-hr assay in the presence of serum to more closely mimic in vivo circumstances. Observed cytolysis was clearly related to TPA concentration and effector cell number. Maximal cytolysis was obtained with TPA at 5 ng/ml, at which specific releases were 43% +/- 6 and 18% +/- 5 (mean +/- 1 SEM) at an effector cell to target cell (E:T) ratio of 2.5:1 and 65% +/- 6, and 41% +/- 12 at an E:T ratio of 20:1, for K562 and U937, respectively. In contrast, unstimulated monocytes expressed minimal cytolytic activity, or at best a low cytotoxic effect at high cellular ratios. When TPA-stimulated monocyte-mediated cytolysis was examined, catalase (2750 U/ml) inhibited K562 and U937 cytolysis by 92% and 84%, respectively; superoxide dismutase (300 U/ml) only inhibited cytotoxicity by 17% and 24%, respectively, implicating a central role of H2O2 rather than superoxide ions. Sodium azide (1 mM), an inhibitor of myeloperoxidase, did not diminish cytolysis; in contrast, it increased K562 and U937 cytolysis by 34% and 57%. This increased cytotoxicity was observed for K562 at low levels of cytotoxicity. These data tend to dismiss an essential role of the H2O2-halide-myeloperoxidase pathway of cytolysis. The OH scavengers, histidine (20 mM) and ethanol (40 mM), did not affect K562 killing; mannitol (50 mM), another OH scavenger, had only a slight inhibitory effect (23%). Finally, H2O2 generated by a glucose-glucose oxidase system directly mediated K562 killing and, to a lesser extent, U937 lysis. These results point strongly towards the role of: 1) a myeloperoxidase-independent mechanism of cytotoxicity, with 2) H2O2 as a key mediator of the cytolytic mechanism, and 3) a limited role of O2.- in synergy with H2O2 in the cytolytic activity of monocytes, and suggest that significant cytolytic function requires an inductive event.     

The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina

The cytotoxic and cytolytic effects of equinatoxin II (EqT II) from the sea anemone Actinia equina L. were studied on exponentially growing and synchronized V-79-379 A cell line in culture. The cell viability test and the determination of the cytolytic effect by cell counting confirmed both cytotoxic and cytolytic activity of EqT II. Additionally, cytocidal and cytostatic effects depending on the toxin concentration were observed. The presence of fetal calf serum in the cell culture medium reduced both cytocidal and cytostatic effects by two magnitudes and prevented cytolysis. Combining EqT II and serum resulted in an insoluble complex which was cytostatic even when isolated and resuspended in the culture medium, while the supernatant retained both cytocidal and cytostatic activity. No significant difference in sensitivity between synchronized and exponentially growing cells could be detected after EqT II treatment.     

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

Further studies on the differences in cytotoxicity of human peripheral blood monocytes and bronchoalveolar macrophages for cultured human lung cells

Summary Previously reported differences between the cytostatic activity of human peripheral blood monocytes (PBM) and bronchoalveolar macrophages (BAM) for cultured human lung tumor cells have been further investigated. The differences are both quantitative and qualitative and are shown not to be due to the respective methods of purification. There was a varying contribution of cytolysis to the cytostasis detected by the ⁷⁵selenomethionine post-labeling assay used. Bronchoalveolar macrophages were cytolytic when tested at both low and high E: T ratios but PBM were only cytolytic at the low E: T ratio. A variable dependence upon soluble cytostatic factor(s) was suggested, and there was evidence of heterogeneity in the factors released by the two populations. Cytostatic factor production by both populations appeared to be under similar regulatory constraints. In vitro maturation of PBM altered their cytostatic dose-response curve to one resembling that previously reported for BAM. It was also shown that sera from poor-prognosis lung tumor patients, which suppressed the in vitro maturation of PBM, also suppressed the in vitro cytostatic activity of PBM for cultured human lung tumor cells.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Effect of human recombinant interferon on cytotoxic activity of natural killer (NK) cells and monocytes

Studies have been performed on the in vitro immunologic effects of homogeneous recombinant human leukocyte interferon, IFLrA. Large granular lymphocytes, enriched for natural killer (NK) cell activity, were pretreated wtih IFLrA or natural interferon preparations and then tested for augmentation of NK activity and of antibody-dependent cell-mediated cytoxicity (ADCC). Monocytes were tested for cytolytic and cytostatic activity in 48–72 hr radioisotopic assays performed in the presence or absence of interferon. Treatment with IFLrA caused significant augmentation of NK, ADCC, and monocyte-mediated cytotoxic activities. Even 10 units of IFLrA induced augmentation of NK activity, and 100 units or more boosted monocyte-mediated activity. The effects in each of these assays were species-specific, with no detectable effects on the activity of mouse effector cells. These results indicate that homogeneous recombinant interferon has potent in vitro immunomodulating effects and thus provide a basis for carefully examining the in vivo effects of this protein on host defenses in forthcoming clinical trials with cancer patients.     

Effect of pyridoxine on tumor necrosis factor activitiesin vitro

Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occuring vitamin B₆ compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line (WEHI 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone WEHI 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agentin vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients.     

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.     

Production and characterization of cytostatic protein factors released from human monocytes during exposure to lipopolysaccharide and muramyl dipeptide

The effect of activating human monocytes in vitro with lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on the production of cytostatic protein factor(s) (CF) has been investigated, and an antiserum against CF has been raised and tested. Upon incubation for 7 hr with LPS, in vitro differentiated human monocytes released CF. During LPS exposure, the presence of the protein synthesis inhibitor cycloheximide, at concentrations which reduced the overall protein synthesis by 60 and 80%, reduced the amount of CF released by only 20 and 40%, respectively. This indicates that the released CF was to a large extent already present in the monocytes before exposure to LPS. Compared to LPS, MDP induced only modest CF release. However, when lymphokine-activated monocytes were exposed to MDP, an increased CF release was observed. By immunizing a rabbit with CF purified by ion-exchange chromatography, chromatofocusing, and gel filtration, an antiserum was raised which neutralized the cytostatic activity released from monocytes exposed to LPS or lymphokines/LPS in sequence on the fourth day of culture. The cytostatic activity obtained by incubating freshly isolated monocytes with LPS was inhibited by the antiserum to a lesser extent, indicating the presence of other cytotoxins or cytotoxic cellular products in addition to CF in supernatants from freshly isolated monocytes. Various CF preparations were tested for IL-1 activity; no correlation between IL-1 activity and cytostatic activity was observed. Moreover, upon gel filtration the CF and IL-1 activities could be separated from each other and are consequently associated with different proteins.