The regulation of pinocytosis in mouse macrophages. I. Metabolic requirements as defined by the use of inhibitors.

A method is described to study the formation of pinocytic vesicles in cultivated mouse macrophages. Vesicles which arise in the peripheral cytoplasm and are in transit to the centrosphere region are enumerated by the phase-contrast microscopy of glutaraldehyde-fixed cells. Under these conditions there is a prompt reversible response of vesicle formation to calf serum factors in the external environment. The reduction of pinosome formation by a variety of metabolic inhibitors was then studied in a medium containing 50% newborn calf serum. Inhibitors of both glycolysis and respiration reduced vesicle formation to low levels. This influence was most striking with cyanide, antimycin A, and anaerobiosis. This indicates an important contribution of an intact respiratory pathway in pinocytosis. Both 2,4-dinitrophenol and oligomycin inhibited vesicle formation at low concentrations. These data suggest a central role of ATP as an energy source for vesicle formation. Inhibitors of protein synthesis, such as puromycin and p-fluorophenylalanine, produced a prompt reduction in vesicle formation. The action of p-fluorophenylalanine was effectively reversed with L-phenylalanine. Actinomycin D depressed pinocytosis to low levels at concentrations of 0.1 to 0.003 microg/ml. This effect was observed only after a 120 min lag phase. A 10 degrees C reduction in ambient temperature lowered vesicle counts to 30% of control preparations at 37 degrees C.

Cholesterol metabolism in the macrophage. II. Alteration of subcellular exchangeable cholesterol compartments and exchange in other cell types.

Macrophage membrane cholesterol is present in two subcellular cholesterol pools, a rapidly exchanging compartment comprising about two-thirds of the total cholesterol, and a slowly exchanging compartment comprising one-third of the total. The morphological identification of the kinetically distinguishable pools proceeded by alteration of each compartment. Trypsin treatment markedly decreased the rate of cholesterol exchange without removing cholesterol from the membrane. Recovery of normal exchange rates took more than 7 hr and required protein synthesis. This suggested that a plasma membrane receptor is involved in positioning of lipoproteins for exchange, and is consistent with the plasma membrane localization of the rapidly exchanging compartment. Extensive pinocytosis by nondegradable dextran, dextran sulfate, or sucrose resulted in the accumulation of many secondary lysosomes, thus increasing the relative proportion of intracellular membranes. The measurable granule membrane area, cholesterol content, phospholipid content, and the relative size of the slowly exchanging cholesterol compartment all increased. The amount of intracellular membrane altered by extensive phagocytosis of latex particles also increased the size of the slowly exchanging cholesterol compartment. This suggested that the slowly exchanging pool of cholesterol represented the intracellular membranes primarily of lysosomal origin. Rabbit alveolar macrophages and thioglycollate-stimulated peritoneal macrophages contain many secondary lysosomes as a result of multiple bouts of in vivo phagocytosis and pinocytosis. In both of these cells the fast and slow pools are equal in size. The increased cholesterol content was attributable to the increase in the relative size of the slowly exchanging compartment. L-cells and melanoma cells also exchange their cholesterol with that of serum lipoproteins. Both cells contain few cholesterol-rich intracellular membranes, and had lower cellular cholesterol contents. In these cells the slowly exchanging pool was a minor contribution to cell cholesterol. Studies with these cells provided further evidence for the lysosomal membrane and plasma membrane localization of the slowly and rapidly exchanging cholesterol compartments.

Cholesterol metabolism in the macrophage. I. The regulation of cholesterol exchange.

The cholesterol metabolism of homogeneous populations of mouse peritoneal macrophages was evaluated under in vitro conditions. Macrophages are rich in free cholesterol and maintain a constant cholesterol to protein ratio (12 microg cholesterol/mg protein). No detectable cholesterol ester was present within the cell. More than 95% of total cholesterol was membrane associated and the majority was present in subcellular fractions containing lysosomes and plasma membrane. Less than 0.1% of cell cholesterol was synthesized from acetate-1-(14)C. During in vitro cultivation, macrophages rapidly exchanged their membrane cholesterol with that of lipoproteins of calf serum. About 30% of the cell cholesterol was exchanged per hour in 20% serum medium, and exchange was nearly complete by 5 hr. Exchange proceeded in a rapid exponential phase followed by a slower phase. Calculations based on a two compartment model indicated that the rapidly exchanging cholesterol compartment represented 60-70% of the total cell cholesterol, and the slowly exchanging compartment accounted for 30-40%. The relationship between serum lipoprotein concentration and exchange rate exhibited first-order kinetics. The rate was determined by thermal energy, in keeping with a Q(10) of 2, and an activation energy of 12 kcal/mole. Exchange was independent of bulk transport of lipoproteins by pinocytosis and phagocytosis, and was not linked to energy metabolism. The alpha-lipoproteins were the major class of proteins of calf serum participating in exchange.

Cholesterol metabolism in the macrophage. 3. Ingestion and intracellular fate of cholesterol and cholesterol esters.

Phagocytosis of cholesterol-containing particles resulted in the formation of an intralysosomal cholesterol compartment. Cholesterol was excreted out of the macrophage with a single exponential rate which depended on the concentration of acceptor lipoproteins in the medium. Exchange kinetics performed on cells which had ingested particulate cholesterol suggested that excretion occurred by the same mechanism as exchange. Cholesterol esters as particulate albumin coacervates were taken up by macrophages and hydrolyzed by a lysosomal cholesterol esterase with optimal activity at pH 4.0. Cholesteryl linoleate was hydrolyzed much more readily than cholesteryl palmitate. The amount of cholesterol esterase and its specific activity increased during the in vitro cultivation of macrophages. Intralysosomally, cholesteryl linoleate and palmitate were hydrolyzed to free cholesterol which was excreted from the macrophage and recovered in the medium. Since cholesteryl linoleate was hydrolyzed more rapidly than free cholesterol was excreted into the medium, free cholesterol accumulated intralysosomally. Cholesteryl palmitate was hydrolyzed more slowly, and the rate of hydrolysis was limiting for excretion of the free cholesterol from within the lysosome.

Trypanosoma cruzi: in vitro induction of macrophage microbicidal activity.

Normal, resident and inflammatory mouse peritoneal macrophages can be induced to display microbicidal activity against trypomastigotes of Trypanosoma cruzi by exposure to products from antigen-pulsed, sensitized spleen cell populations. Optimal macrophage microbicidal activity was achieved by constant exposure and daily renewal of the spleen cell factors. Macrophages obtained after an intraperitoneal injection of mild inflammatory agents were rapidly induced, displaying trypanocidal activity 24 h after exposure to the active spleen cell factor(s), and by 48 h, parasites were no longer observed. Resident peritoneal macrophages required 24 h longer for activation. Removal of the factor(s) before achieving complete disappearance of intracellular parasites led to resumed growth of the surviving organisms. The spleen cell factor(s) is effective when added either before or after exposure of the macrophages to trypomastigotes, and does not itself alter parasite viability. Dilution of the factor(s) up to 1:16 still results in significant trypanocidal activity. In vivo activated cells, obtained after a specific secondary challenge of animals infected with T. cruzi or Bacille Calmette-Guérin, lose their trypanocidal activity under in vitro conditions. This loss of activity can be prevented or restored by the addition of the active spleen cell factor(s). Induction of trypanocidal activity is also obtained with products from Concanavalin A- or lipopolysaccharide-stimulated normal spleen cells.

Bacille Calmette-Guérin infection in the mouse. Regulation of macrophage plasminogen activator by T lymphocytes and specific antigen.

High levels of plasminogen activator (PA) were induced in mouse peritoneal macrophages by infection with BCG, 2-6 X 10(7) viable organisms intravenously, followed 3-4 wk later by intraperitoneal challenge with purified protein derivative (PPD) 2 days before harvest. Macrophages obtained from infected animal without boosting showed little fibrinolytic activity, but challenge of Bacille-Calmette-Guèrin (BCG)-primed peritoneal cells with PPD in culture also enhanced macrophage PA 4- to 10-fold. Stimulation of macrophage PA by PPD depended on specifically sensitized thymus-derived (T) lymphocytes because it was abolished by pretreatment of BCG-primed peritoneal cells with anti-thy 1.2 antiserum and complement. A direct assay was developed in which nylon wool separated sensitized lymphocytes and PPD induced PA in macrophages from uninfected animals under defined conditions on 125I-fibrin. Enhanced macrophage fibrinolysis was proportional to concentration of PPD and the number of sensitized lymphocytes transferred. An indirect two-stage assay was also used to show that BCG-sensitized peritoneal cells released a soluble inducer of macrophage PA into the culture medium, after challenge with PPD. Induction of macrophage PA by PPD challenge in vitro made it possible to study the generation and activity of sensitized peritoneal lymphocytes at different stages of infection. Our results show that nonadherent peritoneal cells of BCG-infected mice provide a rich source of specifically sensitized lymphocytes and that macrophage activation is limited by continued availability of antigen, as well as sensitized lymphocytes. Induction of macrophage PA provides a sensitive, versatile, and rapid in vitro assay to study the role of lymphocytes and specific antigen in macrophage activation by BCG.

The regulation of pinocytosis in mouse macrophages. IV. The immunological induction of pinocytic vesicles, secondary lysosomes, and hydrolytic enzymes.

Bovine sera contain factors which are capable of agglutinating mouse erythrocytes and stimulating the pinocytic activity of cultivated mouse macrophages. The hemagglutinating and vesicle-inducing activities of sera increase with the age of the animal and are absent in fetal calf serum. The majority of this material is recovered in globulin fractions prepared with Na(2)SO(4)-(NH(4))(2)SO(4) and is absent in bovine fraction II. It behaves as a macroglobulin in studies employing zone electrophoresis, Sephadex G-200 filtration, sucrose density gradient centrifugation, and in its sensitivity to 2-mercaptoethanol and heat. Absorption of bovine sera with either mouse erythrocytes or spleen cells removes the hemagglutinating and pinosome-inducing properties of the sera. The addition of small quantities of bovine macroglobulin to mouse macrophages results in a stimulation of pinocytic activity, phase-dense granule formation and the cellular content of three acid hydrolases. In the presence of heat-labile factors, the macroglobulin initiates the hemolysis of mouse erythrocytes and the cytolysis of mouse macrophages. This material is thought to represent an interspecies gammaM-type antibody directed against common antigenic determinants on the mouse erythrocyte and macrophage surface.

The regulation of pinocytosis in mouse macrophages. II. Factors inducing vesicle formation.

The pinocytosis-inducing effect of a number of molecular species was studied in cultures of mouse macrophages. Agents were added to a basal medium containing 1% NBCS-No. 199 and allowed to interact with cells for 150 min. Vesicle counts were then performed and compared to control cells in the basal medium. Certain proteins, i.e. albumin and fetuin, with isoelectric points of five and below were found to be potent stimulators of vesicle formation. Basic proteins including lysozyme, histone, and protamine had little influence at sublethal concentrations. The pinocytosis-stimulating activity of bovine plasma albumin could be markedly depressed by removal of bound fatty acids. The addition of either oleic or linoleic acid to de-fatted albumin restored its inducing properties to initial levels. The activity of fetuin could be abolished by either mild acid hydrolysis or neuraminidase digestion. Both procedures removed the majority of the sialic acid content of fetuin. The D and L isomers of polyglutamic acid were found to produce a marked increase in pinosome production. In contrast, poly-DL-lysine was not effective. Neutral and basic amino acids were without significant effect on pinocytosis, whereas aspartic and glutamic acids were stimulatory. The amides of glutamic and aspartic acid did not induce pinocytosis. The unnatural D isomers of glutamic, aspartic, leucine, and phenylalanine inhibited pinocytosis. The inhibition by D-glutamic acid could be reversed with the L isomer. A number of acid mucopolysaccharides, including heparin, hyaluronic acid, and chondroitin sulfate, were excellent inducers. High molecular weight dextran was without significant stimulatory effect whereas dextran sulfate was very active. Both desoxyribonucleic acid and ribonucleic acid enhanced pinosome formation. A number of low molecular weight anions including N-acetylneuraminic acid were found to enhance vesicle formation. In general, anionic molecules were better inducers than either neutral or cationic species. The minimum effective dose of macroanions was a function of molecular weight and their activity appeared unrelated to specific chemical groupings.

The regulation of pinocytosis in mouse macrophages. 3. The induction of vesicle formation by nucleosides and nucleotides.

A study has been conducted on the influence of nucleosides and nucleotides to induce the formation of pinocytic vesicles in cultured mouse macrophages. Extremely high levels of cytoplasmic vesicles were found after exposure of macrophages to adenosine 5'-phosphate, ADP, and ATP. A limited vesicle response was obtained with adenosine 2'-. and 3'-phosphate, 3',5'-adenosine-phosphate, and deoxyadenosine 5'-phosphate. The di- and triphosphates of guanosine, inosine, cytidine, and uridine were stimulatory but much less active than the adenosine derivatives. Adenosine administration resulted in high levels of pinocytic activity whereas other nucleosides, including inosine, guanosine, cytidine, and uridine, yielded little or no stimulatory effect. Adenosine and its 5'-phosphates produced morphological effects on macrophages characterized by increased spreading, a thin, peripheral cytoplasmic veil with denser cores of oriented mitochondria and contraction of the centre-sphere region. Large numbers of pinosomes were seen in association with mitochondria-containing portions of the cytoplasm. The stimulatory effects of adenosine and ATP were rapid and involved the majority of cells in the culture. Adenosine was unable to reverse the pinocytosis inhibition produced by 2,4-DNP, whereas ATP raised vesicle counts to high levels. Possible mechanisms for these effects are discussed.

The selective inhibition of macrophage phagocytic receptors by anti-membrane antibodies.

Rabbit antibodies were prepared against purified mouse macrophages, erythrocytes, and liver lysosomes. In the presence of complement each of these reagents was capable of lysing mouse erythrocytes and macrophages. In the absence of complement, all antisera agglutinated mouse erythrocytes and at high concentration produced a cytotoxic effect on macrophages. At IgG concentrations of 100 microg/ml, no morphological evidence of cytotoxicity was evident. These data suggest the presence of common antigens on the erythrocyte and macrophage plasma membrane. Anti-macrophage, anti-erythrocyte, and anti-lysosomal gamma-globulins and IgG, employed at subtoxic concentrations, all inhibited the attachment and ingestion of opsonized erythrocytes and mycoplasma. This occurred without significant reduction in the phagocytosis of polystyrene particles, formalinized erythrocytes, and yeast cell walls. Each of the anti-membrane IgG antibodies was capable of blocking the Fc receptor on the macrophage plasma membrane. Attachment to the macrophage membrane occurred by means of the Fab region. However, a role for the Fc portion of the molecule was suggested since pepsin-digested IgG was unable to block the receptor. Each of the IgG antibodies produced a partial blockade of the complement receptor and reduced the ingestion of EAC1,4,2,3 by approximately 50%.

The generation of antigen-specific, major histocompatibility complex-restricted cytotoxic T lymphocytes of the CD4+ phenotype. Enhancement by the cutaneous administration of interleukin 2.

We have examined an in vitro system in which PBMC from purified protein derivative (PPD)-sensitized patients generate CTL after in vitro activation with antigen. These cells selectively destroy mycobacterial antigen PPD-pulsed monocyte targets. These CTL are of the CD4+ phenotype and exhibit MHC class II restriction. After exposure to antigen these cells require 5-7 d for maximal development, whereas, a separate antigen-independent population is generated within 3-4 d. CD8+ cells are poorly, if not at all, cytotoxic under similar conditions. Cells with properties of the NK and LAK lineage are also present in these cultures and kill other specific targets. Human rIL-2 was injected into the skin of lepromatous patients at 10-micrograms doses, given at 48-h intervals, for three doses. Peripheral blood cells obtained 8-14 d after the initiation of IL-2 injection demonstrated enhanced antigen-dependent destruction of monocyte targets. The efficacy of antigen-dependent and -independent populations and their amplification by IL-2-dependent mechanisms is discussed in terms of the local destruction of parasitized macrophages and the subsequent disposal of M. leprae.

Modulation of phagosome-lysosome fusion in mouse macrophages.

A previously described fluorescence assay has been used to characterize factors that modulate phagosome-lysosome (P-L) fusion in mouse macrophages. Fusion was not affected by enzymatic modification or by concanavalin A cross-linking of the plasma membrane or by coating the phagocytic particle with concanavalin A or immune serum. Pretreatment of cells with 10-5-10-4 M colchicine, or treatment immediately after ingestion with 1-10 microgram/ml cytochalasin did not alter P-L fusion; implying that the cytoskeleton does not control fusion in a rate-limiting way. Fusion was strikingly elevated in 5-h cultures of activated macrophages from immune-boosted mice. A lower enhancement was seen in cells activated by proteose-peptone, a nonspecific inflammatory agent.

Phorbol myristate acetate stimulates phagosome-lysosome fusion in mouse macrophages.

The effect of the tumor promoter phorbol myristate acetate (PMA) on phagosome-lysosome (P-L) fusion in mouse macrophages has been studied using a previously described (10) fluorescence assay. Treatment with 0.1--1.0 microgram PMA/ml caused a striking increase in the rate and extent of P-L fusion. Exposure of cells to phorbol, free myristate, or the monoesters of PMA did not reproduce this effect. Macrophages required from 2 to 3 h of pretreatment to express maximal P-L fusion, and this was maintained for at least 20 h when cells were returned to PMA-free medium. Catalase, superoxide dismutase, indomethacin, and hydrocortisone, agents that are known to block the effect of PMA on H2O2, O2-, prostaglandins, or plasminogen activator, did not affect the stimulation of P-L fusion by PMA. The protein-synthesis inhibitors puromycin and cycloheximide did block the PMA effect under conditions in which the high fusion rate of 4-d cells was not affected. Labeled PMA was rapidly taken up by macrophages, with a plateau of uptake at approximately 3 h. When cells were returned to PMA-free medium, cel-associated label was rapidly released, returning to background level within 1 h. The released label was found to be a metabolite of PMA by thin-layer chromatography. This product migrated between the monoester phorbol-12-myristate and free phorbol. Rapid metabolism of PMA was also observed by a macrophage cell line, J774, and, to a lesser extent, by primary rat embryo fibroblasts.

Calcium ionophore synergizes with bacterial lipopolysaccharides in activating macrophage arachidonic acid metabolism.

LPS, a major component of Gram-negative bacterial cell walls, prime macrophages for greatly enhanced arachidonic acid [20:4] metabolism when the cells are subsequently stimulated. The LPS-primed macrophage has been used as a model system in which to study the role of Ca2+ in the regulation of 20:4 metabolism. The Ca2+ ionophore A23187 (0.1 microM) triggered the rapid release of 20:4 metabolites from LPS-primed macrophages but not from cells not previously exposed to LPS. Macrophages required exposure to LPS for at least 40 min before A23187 became effective as a trigger. A23187 (0.1 microM) also synergized with PMA in activating macrophage 20:4 metabolism. The PMA effect could be distinguished from that of LPS since no preincubation with PMA was required. A23187 greatly increased the amount of lipoxygenase products secreted from LPS-primed macrophages, leukotriene C4 synthesis being increased 150-fold. LPS-primed macrophages, partially permeabilized to Ca2+ with A23187, were used to titrate the Ca2+ concentration dependence of the cyclooxygenase and lipoxygenase pathways. Cyclooxygenase metabolites were detected at an order of magnitude lower Ca2+ concentration than were lipoxygenase products. The data suggest that Ca2+ regulates macrophage 20:4 metabolism at two distinct steps: an increase in intracellular Ca2+ regulates the triggering signal and relatively higher Ca2+ concentrations are required for 5-lipoxygenase activity.

The immunological and infectious sequelae of the acquired immune deficiency syndrome.

In spite of much ongoing experimentation, the consensus view is that a vaccine will be difficult to achieve. New strategies of chemo- and immunotherapy may bear more rapid results.

Keratinocyte growth regulation by the products of immune cells.

We describe a bioassay that allows the in vitro investigation of the stimulatory and suppressive factors derived from immune cells in short-term cultures of human keratinocytes. In agreement with other assays, epidermal growth factor is not mitogenic for human keratinocytes. Supernatant fluid from human PBMC stimulated with Con A, from allo-MLRs, as well as supernatants from nonstimulated PBMC, possess growth-promoting molecules. Our results show that both activated and nonactivated T cells release growth factors. Suppressive molecules are produced preferentially by monocyte cultures. Two T cell products, IFN-gamma and transforming growth factor beta are both inhibitory for keratinocyte proliferation. Two other T cell products, IL-3 and GM-CSF, stimulate keratinocyte proliferation at nanogram concentrations. These results suggest the existence of regulatory circuits between the T cells of a dermal inflammatory infiltrate and the overlying epidermal keratinocytes. This may determine the fine control of epidermal proliferation and turnover leading either to enhanced wound repair or skin pathology.

Macrophage oxygen-dependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation.

The capacity of 15 separate populations of mouse peritoneal macrophages to generate and release H2O2 (an index of oxidative metabolism) was compared with their ability to inhibit the intracellular replication of virulent Toxoplasma gondii. Resident macrophages and those elicited by inflammatory agents readily supported toxoplasma multiplication and released 4-20X less H2O2 than macrophages activated in vivo by systemic infection with Bacille Calmette-Guérin or T. gondii, or by immunization with Corynebacterium parvum. Immunologically activated cells consistently displayed both enhanced H2O2 production and antitoxoplasma activity. Exposure to lymphokines generated from cultures of spleen cells from T. gondii immune mice and toxoplasma antigen preserved both the antitoxoplasma activity and the heightened H2O2 release of toxoplasma immune and immune-boosted macrophages, which otherwise were lost after 48-72 h of cultivation. In vitro activation of resident and chemically-elicited cells by 72 h of exposure to mitogen- and antigen-prepared lymphokines, conditions that induce trypanocidal (5) and leishmanicidal activity (14), stimulated O2- and H2O2 release, and enhanced nitroblue tetrazolium reduction in response to toxoplasma ingestion. Such treatment, however, failed to confer any antitoxoplasma activity, indicating that intracellular pathogens may vary in their susceptibility to macrophage microbicidal mechanisms, including specific oxygen intermediates. In contrast, cocultivating normal macrophages with lymphokine plus heart infusion broth for 18H rendered these cells toxoplasmastatic. This in vitro-acquired activity was inhibited by scavengers of O2-, H2O2, OH., and 1O2, demonstrating a role for oxidative metabolites in lymphokine-induced enhancement of macrophage antimicrobial activity. These findings indicate that augmented oxidative metabolism is an consistent marker of macrophage activation, and that oxygen intermediates participate in the resistance of both in vivo- and vitro-activated macrophages toward the intracellular parasite, T. gondii.

The induction of macrophage spreading by factor B of the properdin system.

Unstimulated mouse peritoneal macrophages attached to a glass substratum responded to activated human factor B (Bb) of the properdin system but not to native factor B with rapid spreading and a concomitant increase in their apparent surface area. Excellent correlation of the distribution of Bb protein and cell-spreading activity was found upon purification of Bb by ion-exchange and molecular seive chromatography and alkaline polyacrylamide gel electrophoresis. 1.6 microgram of purified Bb was sufficient to induce spreading in 50% of 5 x 10(4) glass attached macrophages within 1-2 h at 37 degrees C. Treatment of Bb with di-isopropyl-fluorophosphate indicated that the intact catalytic site of the serine-proteinase Bb was required for the initiation of macrophage spreading. The involvement of factor B in the induction of rapid cell spreading could also be indirectly demonstrated in an autologous system in which F(ab')2 fragments of an antiserum to mouse B prevented mouse macrophages from spreading in response to complement-activated mouse serum. These experiments suggest a role for factor B and the alternative pathway of complement fixation in the localization of mononuclear phagocytes to areas of inflammation.

Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates.

A sensitive method for evaluating extracellular parasite viability was used to determine the in vitro susceptibility of virulent Toxoplasma gondii to selected oxygen intermediates. By acridine orange fluorescent staining criteria, toxoplasmas were resistant to up to either 10(-3) M reagent H2O2 or H2O2 generated by glucose-glucose oxidase. In keeping with a lack of sensitivity to H2O2, toxoplasmas contained endogenous catalase (5.7 x 10(-4) Baudhuin units/10(6) organisms). The addition of a peroxidase and halide, however, markedly accelerated killing and lowered the H2O2 requirement by 1,000-fold. In contrast, toxoplasmas were promptly killed after exposure to products generated by xanthine (1.5 x 10(-4) M) and xanthine oxidase (50 micrograms). The inhibition of this system's microbicidal activity by scavengers of O2- (superoxide dismutase) and H2O2 (catalase) indicated that although neither O2- nor H2O2 were toxoplasmacidal, their interaction was required for parasite killing. Quenching OH. and 1O2, presumed products of O2--H2O2 interaction, by mannitol, benzoate, diazabicyclooctane, and histidine, also inhibited toxoplasma killing by xanthine-xanthine oxidase. These findings suggested that O2- and H2O2 functioned in precursor roles and that OH. and 1O2 were toxoplasmacidal. The capacity of normal peritoneal macrophages to pinocytose an oxygen intermediate scavenger, soluble catalase, was also demonstrated. Appreciable extraphagosomal concentrations of catalase were achieved by exposing macrophages to 1 mg/ml of the enzyme for 3 h. Maintenance of high intracellular levels required constant exposure because interiorized catalase was rapidly degraded.

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.