Glutathione depletion sensitizes tumor cells to oxidative cytolysis.
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Animals↗
Biomedical subjects
C F Nathan
Publications and source records attributed to C F Nathan.
Lymphokines enhance the capacity of human monocytes to secret reactive oxygen intermediates.
Supernatants from mitogen- or antigen-stimulated human blood mononuclear cells enhanced the capacity of human monocytes or monocyte-derived macrophages (MDM) to release H(2)O(2) or O(2) in response to phorbol myristate acetate or zymosan. The stimulatory effect of lymphokines (LK) lasted approximately 5 d, regardless of the time of their addition. However, the magnitude of stimulation depended on whether LK were added to freshly explanted monocytes or to MDM. When LK were added on day 0 of culture, they enhanced MDM H(2)O(2)-releasing capacity approximately 40% measured on day 3, when H(2)O(2)-releasing capacity in the controls was maximal. Addition of LK on day 2 retarded the decline in H(2)O(2)-releasing capacity normally seen by day 5, so that LK-treated cells released about twice as much H(2)O(2) as the controls. Addition of LK to MDM that had already lost most of their H(2)O(2)-releasing capacity (e.g., on day 4-6) restored it to an average of 60% of the values seen with freshly explanted monocytes. In this case, LK-treated cells were about 12 times more active than cells incubated in medium alone. The effects of LK were dose- and time-dependent, with maximal effects requiring 3 d of exposure. The specific activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and myeloperoxidase, and the specific content of glutathione were not diminished in LK-treated MDM, suggesting that increased synthesis of H(2)O(2) rather than decreased catabolism probably explained the greater release of H(2)O(2) from LK-treated cells. In contrast, release of H(2)O(2) was suppressed 93+/-4% by exposing monocytes for 4 d to hydrocortisone (50%-inhibitory concentration, 1.9+/-0.3 x 10(-7) M). Thus, the oxidative metabolism of human mononuclear phagocytes can be markedly modulated in vitro: augmented by mediators released from lymphocytes during an immune response, and suppressed by antiinflammatory corticosteroids.
BCG Vaccine↗
Secretion of oxygen intermediates: role in effector functions of activated macrophages.
The ability of macrophages to secrete reactive oxygen intermediates, such as superoxide or hydrogen peroxide, correlates closely with their capacity to kill trypanosoma, toxoplasma, leishmania, and candida. In this sense, secretion of oxygen intermediates is a biochemical marker of macrophage activation. The close correlation between oxidative metabolism and antimicrobial activity appears to stem from the direct involvement of oxygen intermediates in the killing of the same parasites by the macrophages. Similarly, there seem to be at least three experimental settings in which oxygen intermediates play a major role in nonphagocytic lysis of tumor cells by macrophages: in the presence of phorbol myristate acetate, of antitumor antibody, or of a peroxidase derived from eosinophils. These findings direct attention to antioxidant defenses in tumor cells and parasites. The oxidation-reduction cycle of glutathione is one major pathway used by tumor cells to limit oxidative injury by macrophages and granulocytes. Thus, cytotoxicity is augmented by inhibition of glutathione reductase or glutathione peroxidase, by interruption of glutathione synthesis, or by diversion of glutathione into another pathway. On the other hand, catalase appears to play a prominent role in limiting macrophage effector function against toxoplasma.
Animals↗
Role of oxygen in T cell-mediated cytolysis.
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Animals↗
Antitumor effects of hydrogen peroxide in vivo.
Glucose oxidase, covalently coupled to polystyrene microspheres (GOL), produced H(2)0(2) at an average rate of 3.6 nmol/min per 10(9) beads under standard assay conditions. Injection of 1.3 x 10(10) to 1.1 x 10(11) GOL i.p. prolonged the survival of mice by 27 percent after injection of 10(6) P388 lymphoma cells in the same site, consistent with destruction of 97.6 percent of the tumor cells. Placing mice for several hours in 100 percent O(2), the probable rate-limiting substrate for GOL, afforded a 42 percent prolongation of survival from P388 lymphoma, consistent with destruction of 99.6 percent of the tumor cells. When the P388 inoculum was 10(5), 10(4), or 10(3) cells, GOL led to long-term survival (presumed cure) of 23 percent, 77 percent, and 92 percent of the mice, respectively, consistent with reduction of the injected tumor dose to less than 10 cells. Subcutaneous growth of 10(5) P388 cells (approximately 300 lethal dose to 50 percent of mice) was suppressed in 83 percent of mice by admixture of GOL with the tumor cell inoculum. GOL alone had no effect against a more peroxide-resistant tumor, P815 mastocytoma. However, P815 cell glutathione reductase could be inhibited in vivo by well-tolerated doses of the antitumor agent, 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU). BCNU alone cured few mice with P815. Together, BCNU and GOL apparently cured 86 percent of mice injected with 10(6) P815 cells i.p. The protective effect of GOL was abolished by boiling it to inactivate the enzyme, by co-injection of catalase coupled to latex beads, or by delaying the injection of tumor cells for 3 h, by which time the beads had formed aggregates. Soluble glucose oxidase, in doses threefold higher than that bound to GOL, had no detectable antitumor effect. A single injection of preformed H(2)0(2) readily killed P388 cells in the peritoneal cavity, but only at doses nearly lethal to the mice. In contrast, GOL had very little toxicity, as judged by the normal appearance of the mice for over 400 d, gross and microscopic findings at autopsy, and various blood tests. GOL injected i.p. remained in the peritoneal cavity, where it was gradually organized into granulomata by macrophages, without generalized inflammation. Thus, an H(2)0(2)-generating system confined to the tumor bed exerted clear- cut antitumor effects with little toxicity to the host.
Animals↗
Tumor cell anti-oxidant defenses. Inhibition of the glutathione redox cycle enhances macrophage-mediated cytolysis.
The basis of resistance to oxidative injury was studied in six murine tumor cell lines that differed 54-fold in their resistance to enzymatically generated H(2)0(2). The tumors varied 56.7-fold in their specific activity of catalase, 5.3-fold in glutathione peroxidase (GPO), 3.3-fold in glutathione reductase (GR), and 2.7-fold in glutathione. There was no correlation among the levels of the three enzymes, and tumor cell resistance to lysis by H(2)0(2). However, the logarithm of the flux of H(2)0(2) necessary to cause 50 percent lysis of the tumor cells correlated with their content of glutathione (r = 0.91). The protective role of glutathione was analyzed by blocking GR and GPO, the catalysts of the glutathione redox cycle. This was facilitated by the demonstration that the anti-neoplastic agent 1,3-bis-(2- chloroethyl)-l-nitrosourea (BCNU) was a potent inhibitor of GR in intact tumor cells. BCNU inactivated tumor cell GR with a 50 percent inhibitory dose of 11 muM and a t(l/2) of inhibition of 30 s. Complete inhibition of GR was attained with no effect on GPO or catalase. Tumor cells whose GR was inactivated by BCNU could be lysed by fluxes of H(2)0(2) to which they were otherwise completely resistant. They could be killed by phorbol myristate acetate (PMA)-stimulated, bacilli Calmette-Guerin-activated macrophages in numbers which were otherwise insufficient, and by nonactivated macrophages, which otherwise were ineffective. BCNU-treated target cells were also much more sensitive to antibody-dependent, macrophage-mediated cytolysis. However, such tumor cells were no more sensitive than controls to lysis by alloreactive T cells or by antibody plus complement. Next, we deprived tumor cells of selenium by passage in selenium-deficient mice. GPO was inhibited 85 percent in such cells, with no effect on GR or catalase. Tumor cells with reduced GPO activity were markedly sensitized to lysis by small fluxes of H(2)0(2) or by PMA-stimulated macrophages or granulocytes. In contrast, inhibition of catalase with aminotriazole had no effect on the sensitivity of three tumors to peroxide-mediated lysis, and had modest effects with two others. Thus, the oxidation-reduction cycle of glutathione serves as one of the major defense mechanisms of tumor cells against three related forms of oxidant injury: lysis by fluxes of H(2)0(2), by PMA-triggered macrophages, and by macrophages in the presence of anti-tumor antibody.
Animals↗
Hydrogen peroxide metabolism in human monocytes during differentiation in vitro.
The capacity of human blood monocytes to secrete hydrogen peroxide (H2O2) and superoxide (O2-) was measured as the cells differentiated during 4 wk of culture. Morphologic transformation of monocytes into macrophages, epithelioid cells, and multinucleated giant cells accompanied a steady increase in the content of protein per cell, from 0.77 mg/10(7) cells on days 0 to 11.77 mg/10(7) cells on days 20 to 29. In contrast, secretion of H2O2 by adherent monocytes was 859 +/- 73 nmol/60 min per mg protein (mean +/- SEM, n = 18) on day 0, rose 40% on day 3, and then fell rapidly, remaining below 6% of the initial values after day 10. The decline in capacity to secrete reactive oxygen intermediates was observed whether H2O2 or O2- were measured, whether the cells were challenged with phorbol myristate acetate or with opsonized zymosan, and whether the results were expressed per milligram cell protein or per cell. Superoxide dismutase activity tripled in adherent monocytes from day 0 to day 3, and thereafter remained elevated through at least day 16. In contrast, the activity of myeloperoxidase declined rapidly, catalase and glutathione peroxidase declined more gradually, and glutathione reductase and glutathione remained constant through the period of observation. Thus, the decline in capacity to secrete H2O2 could not be attributed to increases in cellular levels of these antioxidants. On the first day of culture, H2O2 release was enhanced up to fourfold by inclusion of sodium azide or potassium cyanide in the assay medium. This enhancement appeared to be due to inhibition of monocyte myeloperoxidase, rather than catalase. This conclusion was based on the kinetics and dose-response relationships for the effects of azide and cyanide on H2O2 release and on the activities of catalase and myeloperoxidase. Thus, the differentiation of human monocytes into macrophages in vitro is accompanied by an apparent reduction in the capacity to produce H2O2 and O2-. In this regard, the human monocyte-derived macrophage comes to resemble the resting tissue macrophage previously characterized in the mouse peritoneal cavity.
Cell Adhesion↗
Hydrogen peroxide and cytolytic factor can interact synergistically in effecting cytolysis of neoplastic targets.
Two secretory products from activated macrophages, which are thought to be involved in the lysis of tumor cells, are H2O2 and cytolytic factor (CF). To examine the potential of interaction between these 2 mediators of cytolysis, we first exposed MCA-I sarcoma targets to the 2 lytic agents. MCA-I targets, which have been previously shown to be lysed by CF, could also be lysed by H2O2 (the concentration of H2O2 causing 50% lysis of targets was approximately 1.4 x 10(-3) M). Exposure of the MCA-I targets to a nonlytic concentration of H2O2 for 1 hr potentiated the subsequent lytic effects of CF and did so synergistically. Exposure of targets to CF, however, did not potentiate the subsequent lytic effects of H2O2, even when the targets were treated with a lytic concentration of CF for 4 hr. Similar results were seen with P-388 lymphoma targets. The P388 cells were quite sensitive to the lytic effects of H2O2 (the concentration of H2O2 causing 50% lysis of these targets was approximately 2.0 x 10(-5) M) but proved relatively resistant to CF in comparison to the MCA-I targets. The data suggest that H2O2 and CF can interact synergistically to produce cytolysis of neoplastic targets if the targets are exposed to the 2 substances in a defined sequence.
Animals↗
Macrophage oxygen-dependent antimicrobial activity. IV. Role of endogenous scavengers of oxygen intermediates.
The activities of the endogenous O2- and H2O2 scavenging enzymes, superoxide dismutase (SOD), glutathionine peroxidase (GP), and catalase, were measured in lysates of the intracellular parasite, Toxoplasma gondii, and in various macrophage populations. During 72 h of cultivation in standard medium alone, the catalase activity of in vivo-activated toxoplasma-immune macrophages (IM) and immune-boosted macrophages (IB) progressively increased by eight- to ninefold, and correlated with the previously observed parallel decline in these cells' antitoxoplasma activity and capacity to release H2O2. SOD and GP activities either remained constant or decreased during this 3-d period. Lymphokine exposure, which preserved the antitoxoplasma activity and oxidative capacity of 48- and 72-h cultures of IB and IM cells, blunted the rise in catalase levels and had no effect on SOD or GP. Inhibition of IB and IM macrophage catalase by aminotriazole maintained toxoplasmastatic activity otherwise lost after 48 h of cultivation. In addition, IB and IM cells from acatalasemic mice contained 20- to 30-fold less catalase, and showed comparatively little decline in either H2O2 release or antitoxoplasma activity during 72 h in culture. In vitro-(lymphokine) activated resident macrophages from normal mice had the highest levels of SOD, GP, and catalase, and these cells failed to kill or inhibit T. gondii despite enhanced extracellular release of O2- and H2O2. Toxoplasmas were also found to contain all three enzymatic scavengers. Aminotriazole inhibition of lymphokine-activated cells' catalase or of toxoplasma catalase was effective in inducing these macrophages to display antitoxoplasma activity. Moreover, and in contrast to normocatalasemic resident cells, those from acatalesemic mice were readily induced by lymphokine to inhibit the replication of untreated virulent toxoplasmas. These results suggest that endogenous O2- and H2O2 scavenging enzymes, which function within both T. gondii and activated macrophages as host cell antioxidant protective mechanisms, may reduce the effectiveness of phagocyte antimicrobial activity. Thus, the presence of SOD, GP, and especially catalase within both target and effector cell may be important determinants of macrophage oxygen-dependent processes.
Animals↗
The macrophage as an effector cell.
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Antigen-Antibody Complex↗
Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates.
The capacity of three populations of mouse peritoneal macrophages to generate oxidative metabolites (as judged by extracellular release of H2O2) was compared to their ability to influence the intracellular fate of virulent Toxoplasma gondii. Macrophages from normal mice released little H2O2 and allowed unrestricted multiplication of intracellular toxoplasmas. Cells from chronically infected, immune (IM) mice released 4 times more H2O2 and displayed microbistatic activity. In contrast, macrophages from immune-boosted (IB) mice released 25 times more H2O2 than normal cells and rapidly killed the bulk of ingested toxoplasmas within 1 h. When macrophage monolayers were exposed to scavengers of O2-, H2O2, OH., and 1O2, both the inhibition of intracellular toxoplasma multiplication by IM macrophages and the killing of toxoplasmas by IB macrophages were reversed. Depriving cells of glucose, which markedly reduced H2O2 release, resulted in similar reversal of IM and IB macrophage anti-toxoplasma activity. As judged by the effect of the individual oxygen intermediate scavengers, O2- and H2O2 appeared to serve as precursors for the key toxic agents which may include OH. and 1O2. Providing normal macrophages with an exogenous source of oxidative metabolites generated by xanthine and xanthine oxidase, but not glucose and glucose oxidase, resulted in inhibition of intracellular toxoplasma growth. These findings suggest the presence of an oxygen-dependent antimicrobial system in mononuclear phagocytes beyond the production of O2- and H2O2, and indicate an important role for oxygen intermediates in macrophage resistance to the intracellular pathogen T. gondii.
Animals↗
Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity.
When deprived of oxygen, Bacille Calmette-Guérin (BCG)-activated macrophages no longer lysed P388 lymphoma cells. Both H2O2 release and cytotoxicity by BCG-activated macrophages and by granulocytes triggered with phorbol myristate acetate (PMA) were markedly inhibited when the glucose concentration in the medium was reduced to 0.03 mM or less, or if glucose were replaced with galactose. Catalase abolished PMA-triggered cytotoxicity by both types of effector cells, whereas superoxide dismutase had no effect. Ferricytochrome C reduced the cytotoxicity of BCG-activated macrophages, an effect which was largely reversed by superoxide dismutase. 10 drugs, thought to quench singlet oxygen and/or scavenge hydroxyl radical, did not affect cytotoxicity in this system. Neither azide nor cyanide reduced cytolysis, but there was marked inhibition by lactoperoxidase and iodide. This suggested that cytotoxicity was not dependent upon myeloperoxidase, and that lactoperoxidase may have diverted H2O2 from the oxidation of target cells to oxidation of substances in serum. Mouse erythrocytes, although sensitive targets, interfered with the cytolysis of lymphoma cells, probably by competition for H2O2. Starch particles with covalently bound glucose oxidase resembled macrophages in their spatial relation to the target cells and in the flux of H2O2 they generated from their surface, but were not expected to produce any other potentially toxic products. Such particles lysed lymphoma cells, and the lysis was prevented by catalase. Neither arginase nor thymidine appeared to be involved in cytolysis by BCG-activated macrophages under the conditions used. These findings demonstrated that release of H2O2 was both necessary and sufficient for cytolysis by BCG-activated macrophages and by granulocytes when pharmacologically triggered.
Animals↗
Extracellular cytolysis by activated macrophages and granulocytes. I. Pharmacologic triggering of effector cells and the release of hydrogen peroxide.
Lymphoma cells were rapidly lysed by activated macrophages and granulocytes in the presence of PMA. Release of 51Cr from lymphoma cells correlated closely with their destruction as viewed by scanning electron microscopy, and with reduction in the number of trypan blue-excluding cells. The standard assay involved 51 Cr release measured at 4.5 h, but injury appeared to be complete in 1 h. Of eight different types of effector cells tested, only those releasing abundant H2O2 in response to PMA were effective, that, is BCG-, C. parvum-, or casein-activated macrophages, or thioglycollate-elicited granulocytes. Normal macrophages, J774 cells, or macrophages elicited with thioglycollate broth or proteose-peptone were ineffective. BCG-activated macrophages and granulocytes caused 50% specific release of 51Cr from P388 lymphoma cells at E:T ratios between 1.4 and 4.5, and from mouse erythrocytes at E:T ratios of 0.017 to 0.025. 10 types of target cells varied widely in their susceptibility to lysis by reagent H2O2, with one-half maximal lysis occurring at H2O2 concentrations ranging from 3.63 X 10(-6) M to 3.85 X 10(-5) M. Effector cells were expected to generate approximately that much H2O2 during the period of injury. Susceptibility of the target cells to lysis by PMA-triggered granulocytes correlated closely with their sensitivity to H2O2 (r = 0.98). The membrane-active agents LPS and digitonin, which did not trigger H2O2 release, did not trigger cytotoxicity. The dose-response curve for triggering of H2O2 release by PMA was identical to that for triggering cytotoxicity. These results provided strong circumstantial evidence for the importance of H2O2 in extracellular cytolysis by activated macrophages and granulocytes when pharmacologically triggered.
Animals↗
Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering.
Using a specific and sensitive fluorometric assay, the H2O2 release from as few as 2 X 10(5) mouse peritoneal macrophages could be detected continuously and quantitated. It is emphasized that the assay measured H2O2 release, not production. Induction of H2O2 release required sequential application of two stimuli: the administration of an activating agent to the mice from 4 days to 10 wk before all harvest, and the exposure of the cells in vitro to a triggering agent. BCG was most effective as an activating agent, resulting in peritoneal macrophages which could be triggered to release H2O2 almost as copiously (8 nmol/10(6) macrophages per 5 min) as mouse peritoneal PMN (9 NMOL/10(6) PMN per 5 min). Casein and C. parvum could also serve as activators, but thioglycollate and FCS were ineffective after single injections. PMA was a potent triggering agent, resulting in a maximal rate of H2O2 release after a latency of about 40 s for cells in suspension. Other triggering agents included the ionophore A23187, concanavalin A in the presence of cytochalasin B, and phagocytosis. H2O2 release could be attributed to macrophages and PMN in peritoneal cell suspensions or in preparations of adherent peritoneal cells, but not to lymphocytes. Indirect evidence suggested that the H2O2 detected was formed from superoxide anion. These observations appear to justify renewed interest in the idea that H2O2 may be important in macrohpage antimicrobial and antitumor mechanisms.
Animals↗
Decreased phagocytosis by peritoneal macrophages from BCG-treated mice: induction of the phagocytic defect in normal macrophages with BCG in vitro.
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Animals↗
Characterization of the nonphagocytic adherent cell from the peritoneal cavity of normal and BCG-treated mice.
A distinctive subpopulation of nonphagocytic, tightly adherent cells (NPAC) comprised approximately 6% of the adherent peritoneal cells from untreated mice, and about 18% of those from mice previously given BCG i.p. A separation procedure based on adherence and lack of phagocytosis was devised. Isolated NPAC were morphologically intermediate between small lymphocytes and macrophages. They were positive for nonspecific esterase, negative for peroxidase, positive for surface IgM, and negative for surface IgG1, IgG2 and IgA. When capped, their surface IgM regenerated in vitro. NPAC had demonstrable Fc receptors but not EAC receptors. They resisted killing by an anti-macrophage serum, were negative by immunofluorescence with an anti-T cell reagent, and incorporated increased amounts of thymidine in response to LPS but not to PHA. They were more readily killed with anti-Ia serum and complement than macrophages, but less readily than splenic B cells. NPAC appeared to represent a subpopulation of B lymphocytes which contaminates some preparations previously regarded as "macrophages" and which may be ressponsible for some of the activities previously ascribed to "macrophages".
Animals↗
Isolation of a subpopulation of adherent peritoneal cells with anti-tumour activity.
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Animals↗
Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages.
Peritoneal macrophages from mice infected with Bacille Calmette-Guérin (BCG) and from normal mice were examined for their effects in vitro on thymidine uptake by 10 murine lymphomas, a murine fibroblast line, and a guinea pig hepatoma. Only the murine fibroblast line showed growth inhibition in the presence of BCG macrophages. For the majority of tumors, normal macrophages were profoundly stimulatory to tumor cell DNA synthesis, while BCG macrophages were much less stimulatory, without being frankly inhibitory. The effect of 2-mercaptoethanol on tumor cell growth was also studied. All lymphomas stimulated to grow more rapidly in vitro by normal macrophages were stimulated to a similar degree by 2-mercaptoethanol.
Animals↗