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.

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.

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.

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. 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.

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 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. 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.

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.

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.

Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis: evidence for a two-stage process.

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1-2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated gamma-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

Effects of concanavalin A on mouse peritoneal macrophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation.

Concanavalin A (Con A) binds to saccharide residues on the mouse peritoneal macrophage plasma membrane and stimulates extensive pinocytic interiorization of the membrane. The overall pinocytic rate is increased 3.5-4.5 times by the addition of Con A, and the surface marker enzyme adenosine triphosphatase can be identified histochemically in association with the cytoplasmic vesicles generated after exposure of the cells to Con A. Once formed, these pinocytic vesicles may persist for several days and fail to show morphologic evidence of fusion with primary or preformed secondary lysosomes. There is no apparent effect on the capacity of the macrophage to ingest either latex particles or IgG-coated SRBC administered either simultaneously with or subsequent to the Con A.

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.

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 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.

The induction of macrophage spreading: role of coagulation factors and the complement system.

Unstimulated mouse peritoneal macrophages, attached to either glass or plastic substrates, responded to factors generated in serum and plasma by spreading and increasing their apparent surface area up to eightfold. Two distinct and dissociable systems were involved. The first appears related to the distinct and dissociable systems were involved. The first appears related to the contact phase of blood coagulation. It is activated by glass and not plastic surfaces, depleted by kaolin adsorption, and inhibited by soybean trypsin inhibitor. In contrast, a separate complement-dependent system can be generated in kaolin-adsorbed plasma. Activation of the complement system can occur either by the alternate or classical pathways and generates a relatively small effector molecule which is dialyzable. These factors presumably influencing the surface membrane and underlying structures may explain the rapid spreading of activated macrophages observed after both infections and chemical peritoneal inflammatory agents.

5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations.

Mouse resident peritoneal macrophages display sufficient 5'-nucleotidase activity to hydrolyze 58 nm AMP/min per cell protein. This activity increases approximately 163 nm AMP/min per mg after 72 h in culture. The enzyme is renewed in unstimulated cells with a half-time of 13.9 h. The activity is not reduced by treatment of intact cells with a variety of proteolytic enzymes, including trypsin, pronase, urokinase, and plasmin. Cells obtained from an inflammatory exudate have diminished or absent levels of enzyme activity. Endotoxin-elicited cells display enzyme activitiy of 20.9 nm AMP/min per mg, while thioglycollate-stimulated macrophages have no detectable activity. The reduced level of activity in endotoxin-stimulated cells is due to their elevated rate of enzyme degradation, with a half-time of 6.9 h. Their rate of enzyme synthesis is essentially normal. No evidence for latent enzyme activity could be obtained in thioglycollate-stimulated cells, nor do these cells produce any inhibition of normal cell enzyme activity. Serum deprivation reduces the enzyme activity of resident cells to about 45% of control activity. These conditions do not significantly affect the rate of enzyme synthesis, but again are explainable by an increase in the rate of enzyme degradation. Pinocytic rate is elevated in endotoxin-stimulated cells which show a more rapid rate of enzyme degradation than unstimulated cells do. However, in serum-free conditions, the rate of enzyme degradation is doubled with no change in the pinocytic rate of the cells.

5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis.

The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface.

In vitro induction of lysosomal enzymes by phagocytosis.

The in vitro induction of lysosomal enzymes by phagocytosis was demonstrated in cultivated mouse peritoneal macrophages. The contribution of each of several steps in the endocytic process to enzyme induction was examined. The enzymatic response after the uptake of equal numbers of erythrocytes (RBC) and nondigestible particles were compared. Phagocytosis of RBC produced a marked increase in the levels of acid phosphatase, beta-glucuronidase, and cathepsin D. Puromycin (1 microg/ml) inhibited the enzyme response. In contrast, phagocytosis of polyvinyl toluene, polystyrene, and insoluble starch particles produced no increase in macrophage lysosomal enzymes, although fusion of phagosomes with preexisting lysosomes occurred normally. The endocytic stimulus to synthesis of inducible lysosomal enzymes, therefore, occurred at or beyond the stage of digestion. Purified protein (bovine gamma globulin) aggregates and homopolymer coacervates of poly-l-glutamic acid: poly-l-lysine were effective inducers of lysosomal acid phosphatase, beta-glucuronidase, and cathepsin D, whereas homopolymers of the same D-amino acids were ineffective as inducers. Both the quantity of phagocytized substrate and its rate of enzymatic hydrolysis appear to control the level and persistance of lysosomal hydrolases.