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.

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.

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.

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.

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.

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'-.

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.

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 in vitro differentiation of mononuclear phagocytes. V. The formation of macrophage lysosomes.

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles.

The in vitro differentiation of mononuclear phagocytes. IV. The ultrastructure of macrophage differentiation in the peritoneal cavity and in culture.

The structure of unstimulated mouse peritoneal phagocytes has been examined by electron microscopy and compared to cells obtained from the inflamed peritoneum and from cultures maintained in vitro. The unstimulated cell resembles the blood monocyte and contains a moderate amount of rough surfaced endoplasmic reticulum, a small but well defined Golgi apparatus and a few, small, electron-opaque granules in the cytoplasm. During in vitro cultivation there are marked changes in cell ultrastructure.

The isolation and selected properties of blood monocytes.

A technique is described for the quantitative recovery of monocytes from horse blood by means of flotation on dense albumin solutions. Monocytes are concentrated in a surface pellicle along with a few lymphocytes which are then removed when the monocytes adhere to a glass surface. The in vitro cultivation of homogeneous populations of monocytes results in an increase in (a) cell size, (b) number of mitochondria, and (c) phase-dense granules of the centrosphere.

The in vitro differentiation of mononuclear phagocytes. 3. The reversibility of granule and hydrolytic enzyme formation and the turnover of granule constituents.

Mouse mononuclear phagocytes cultivated in 50 per cent newborn calf serum medium pinocytize actively and form large numbers of phase-dense granules as well as three hydrolytic enzymes. When such cells are then placed in 1 per cent newborn calf serum they illustrate (a) a low level of pinocytic activity, (b) a shrinkage in granule size, and (c) a loss in cell protein, acid phosphatase, beta-glucuronidase, and cathepsin. Examination of the extracellular medium revealed no detectable hydrolase activity.

THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES. II. THE INFLUENCE OF SERUM ON GRANULE FORMATION, HYDROLASE PRODUCTION, AND PINOCYTOSIS.

The concentration of newborn calf serum in the medium has marked effects on the morphological and biochemical properties of mouse mononuclear phagocytes. At a low serum concentration, the cells developed small numbers of tiny cytoplasmic granules and little or no increase in acid phosphatase, cathepsin, and beta-glucuronidase. As the serum concentration was raised, granules were formed at a more rapid rate and were larger in size.

THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES. I. THE INFLUENCE OF INHIBITORS AND THE RESULTS OF AUTORADIOGRAPHY.

The influence of selected inhibitors of protein synthesis on the in vitro differentiation of mouse mononuclear phagocytes has been investigated. DL-p-Fluorophenylalanine at concentrations of 250 microg/ml inhibits the formation of three lysosomal hydrolases, cytochemically demonstrable acid phosphatase and osmiophilic, phase-dense granules. These effects occur in the absence of cell death and are reversed by L-phenylalanine.

THE DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES. MORPHOLOGY, CYTOCHEMISTRY, AND BIOCHEMISTRY.

The in vitro differentiation of homogeneous populations of monocyte-like cells from the unstimulated mouse peritoneal cavity is described. Under the conditions employed, a progressive increase in cell size occurs without significant cell division. This process is characterized morphologically by the accumulation of phase-dense and neutral red-positive granules, mitochondria, and lipid droplets.

THE FATE OF BACTERIA WITHIN PHAGOCYTIC CELLS. 3. DESTRUCTION OF AN ESCHERICHIA COLI AGGLUTINOGEN WITHIN POLYMORPHONUCLEAR LEUCOCYTES AND MACROPHAGES.

The fate of a heat-stable Escherichia coli agglutinogen within three types of rabbit phagocytic cells was examined. A system is described whereby quantitative ingestion of viable E. coli by suspensions of PMN leucocytes, BCG-induced alveolar macrophages, and oil-induced peritoneal macrophages took place in vitro.

THE PARTICULATE HYDROLASES OF MACROPHAGES. I. COMPARATIVE ENZYMOLOGY, ISOLATION, AND PROPERTIES.

The contents of selected hydrolytic enzymes of oil-induced peritoneal, normal alveolar, and BCG-induced alveolar macrophages have been studied. On a per cell or nitrogen basis the normal alveolar cells contained considerably more acid phosphatase, cathepsin, acid ribonuclease, lysozyme, and lipase than peritoneal cells. The BCG-induced alveolar macrophage exhibited increased levels of acid phosphatase, lysozyme, and lipase as compared to alveolar macrophages from unstimulated rabbits.

THE PARTICULATE HYDROLASES OF MACROPHAGES. II. BIOCHEMICAL AND MORPHOLOGICAL RESPONSE TO PARTICLE INGESTION.

The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme.