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Biomedical subjects

Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 217 records · Page 12Linked to original sources
PubMed1972

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.

Acid Phosphatase↗
PubMed1972

The mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells.

There have been many attempts in the past to classify phagocytic mononuclear cells and to define the cell system they are considered to form-among these being the "macrophage system" of Metchnikoff, the "reticulo-endothelial system" of Aschoff, and the "reticulo-histiocyte system" proposed by Volterra and reintroduced by Thomas. None of these is entirely adequate in the light of present knowledge. In 1969, therefore, a group of workers proposed a new classification of all highly phagocytic mononuclear cells and their precursors in what they termed the "mononuclear phagocyte system". This system includes the promonocytes and their precursors in the bone marrow, the monocytes in the peripheral blood, and the macrophages in the tissues. Subsequent consultation with numerous other specialists throughout the world led to a certain number of changes in this classification, which is now proposed in revised form.Inclusion of cells in the "mononuclear phagocyte system" is based on similarities in the morphology, function, origin, and kinetics of the phagocytes. By these criteria reticular cells, dendritic cells, endothelial cells, and fibroblasts (fibrocytes) are excluded. The proponents point out that as new knowledge is acquired modifications may have to be made, certain cells being added to or removed from the new classification.

Bone Marrow Cells↗
PubMed1971

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.

Acid Phosphatase↗
PubMed1971

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.

Animals↗
PubMed1970

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.

Acid Phosphatase↗
PubMed1969

The uptake, storage, and intracellular hydrolysis of carbohydrates by macrophages.

The exposure of cultivated mouse macrophages to sucrose (0.009-0.03 M) leads to the formation of large phase- and electron-lucent, acid phosphatase-positive vacuoles in the perinuclear region. The vacuolization process and the uptake of sucrose-(14)C is blocked by inhibitors of pinocytosis and stimulated by calf serum in the medium. These results suggest the uptake of sucrose by pinocytosis and its subsequent segregation and storage in secondary lysosomes. The addition of sucrose also increases the total content of three macrophage lysosomal hydrolases. The addition of invertase to the environment of sucrose-laden macrophages leads to the prompt shrinkage of the sucrose-containing lysosomes. This is accompanied by the intracellular hydrolysis of sucrose to fructose and glucose residues which are promptly excreted into the medium. The uptake of invertase, as indicated by the shrinkage of sucrose-containing vacuoles, is blocked by inhibitors of pinocytosis. No effect was noted when invertase was added to macrophages laden with Ficoll, a polysucrose which is not hydrolyzed by the enzyme. The influence of other carbohydrates was then investigated. Monosaccharides with molecular weights up to 220 did not produce vacuolization. However, a certain number of di-, tri-, and tetrasaccharides produced vacuolization identical with that of sucrose. Each of the disaccharides which produced vacuolization was resistant to the complement of macrophage hexosidases, whereas those that were ineffective were degraded by either macrophage or serum enzymes. The addition of beta-glucosidase to cellobiose-laden macrophages resulted in the shrinkage of vacuoles but did not alter the vacuoles of sucrose containing cells. The ability of small, neutral carbohydrates to produce lysosomal swelling is dependent upon both molecular weight and their resistance to lysosomal hydrolases.

Acid Phosphatase↗
PubMed1969

The fate of peptides pinocytosed by macrophages in vitro.

A series of small peptides, such as might arise in the course of intralysosomal protein digestion, were screened for the ability to escape, intact, from mouse peritoneal macrophage lysosomes. Inability to penetrate lysosomal membranes was inferred from a peptide's induction of lysosomal swelling, or vacuolization, in cultured macrophages. Two of the peptides tested, (D-Glu)(2) and (D-Ala)(3), induced vacuolization. Neither peptide was susceptible to hydrolysis by enzymes in macrophages or in the serum-containing culture medium. Their morphological effect was inhibited by parafluorophenylalanine, an inhibitor of pinocytosis. Once formed by either peptide, the vacuoles persisted for several hours in peptide-free medium. Quantitative studies of radioactively labeled (D-Glu)(2) confirmed the morphological evidence that (D-Glu)(2) is taken up by pinocytosis and stored, intact, in macrophage lysosomes. The majority of the peptides which failed to induce vacuolization-(L-Ala)(2), L-Ser.L-Ala, L-Val.L-Ala, L-Ala.L-Thr, Gly.D, L-Phe, L-Ala.D-His, (L-Ala)(3), (L-Glu)(2), and D-Leu.L-Tyr-were found to be susceptible to hydrolysis by cellular or serum peptidases. Their failure to induce vacuolization was attributed to their hydrolysis to subunits capable of penetrating lysosomal membranes. Some of the peptides which had failed to induce vacuolization-(D-Ala)(2), D-Ser.D-Ala, D-Val.D-Ala, Gly-D-Asn, D-Ala.D-Thr, and D-Arg.D-Val-were found to be indigestible. Except for the cytotoxic peptide D-Arg.D-Val, peptides in this category all had lower molecular weights and volumes than (Glu)(2) or (Ala)(3). It is inferred that these peptides are small enough to escape from macrophage lysosomes, while (Glu)(2) and (Ala)(3) are too large to escape intact. The implications of this inference for the mechanism of intracellular digestion of pinocytosed proteins are discussed.

Alanine↗
PubMed1968

The origin and kinetics of mononuclear phagocytes.

The origin and turnover of efferent populations of mouse mononuclear phagocytes has been described. Mononuclear phagocytes were defined as mononuclear cells which are able to adhere to glass and phagocytize. In vitro labeling studies with thymidine-(3)H showed that monocytes in the peripheral blood and peritoneal macrophages do not multiply and can be considered end cells in a normal, steady state situation. However, the mononuclear phagocytes of the bone marrow appear to be rapidly dividing cells. This conclusion was supported by in vivo labeling experiments. A peak of labeled mononuclear phagocytes of the bone marrow was found 24 hr after a pulse of thymidine-(3)H. This was followed, 24 hr later, by a peak of labeled monocytes in the peripheral blood. From these experiments it was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes. Labeling studies after splenectomy and after X-irradiation excluded other organs as a major source of the monocytes. Peak labeling of both the blood monocyte and peritoneal macrophages occurred at the same time. A rapid entry of monocytes from the blood into the peritoneal cavity was observed, after a sterile inflammation was evoked by an injection of newborn calf serum. These data have led to the conclusion that monocytes give rise to peritoneal macrophages. No indications have been obtained that mononuclear phagocytes originate from lymphocytes. In the normal steady state the monocytes leave the circulation by a random process, with a half-time of 22 hr. The average blood transit time of the monocytes has been calculated to be 32 hr. The turnover rate of peritoneal macrophages was low and estimated at about 0.1% per hour. On the basis of these studies the life history of mouse mononuclear phagocytes was formulated to be: promonocytes in the bone marrow, --> monocytes in the peripheral blood, --> macrophages in the tissue.

Animals↗
PubMed1968

Autophagic vacuoles produced in vitro. I. Studies on cultured macrophages exposed to chloroquine.

Mouse macrophages exposed to 30 microg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1-4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.

Acid Phosphatase↗
PubMed1968

Autophagic vacuoles produced in vitro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine.

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1-2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.

Animals↗
PubMed1967

The uptake and digestion of iodinated human serum albumin by macrophages in vitro.

Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate formation of pinocytic vesicles. Autoradiography of cells pulsed with (125)I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of (125)I-HSA, intracellular radioactivity decreases and the amount of TCA-soluble isotope in the medium increases correspondingly. About 50% of the intracellular isotope is lost in 5 hr. The release of isotope from pulsed cells is not inhibited by parafluorophenylalanine, 2,4-dinitrophenol or by a reduction of the serum concentration of the medium. However, the processing of ingested (125)I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.

Animals↗
PubMed1967

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.

Agglutination↗