Showing papers on "Phagosome published in 1980"

Ammonia inhibits phagosome–lysosome fusion in macrophages

Abstract: When foreign bodies, including many microorganisms, are ingested by cultured macrophages, they become enclosed in phagosomes, with which lysosomes usually fuse and then discharge their enzymes and other contents into the resulting phagolysosomes. Such fusion is, however, diminished or absent after the phagocytosis of some pathogens, notably Mycobacterium tuberculosis1 and Toxoplasma gondii2. Assuming that the nonfusion is due to active inhibition by the intraphagosomal microbe, identification of an inhibitor should clarify the lysosomal control mechanism. It has been suggested that strongly acidic sulphatides present in virulent tuberculosis, which, like other substances with polyanionic structural features, can themselves block phagosome–lysosome fusion (P-LF)3–5, may contribute to the negative lysosome response to ingested tubercle bacilli4. We report here another possibility, based on inhibition of fusion of yeast-containing phagosomes by filtrates from cultures of tubercle bacilli on traditional-type defined media; we show that the ammonia content of such filtrates is sufficient to account for their effect. This inhibition of fusion seems to be an hitherto unrecognized intracellular consequence of added ammonia, in striking contrast to the enhancement produced by some lipophilic amines3.

Characterization of a human macrophage-like cell line stimulated in vitro: a model of macrophage functions.

Abstract: When cells of human macrophage-like cell line U937 are cultured in the presence of medium conditioned by mixed lymphocyte culture (MLC), PHA- or Con A-stimulated lymphocytes they demonstrate morphologic and functional characteristics of stimulation The cells become larger, their surface more villous, and the cytoplasm has increased numbers of lysosomes and phagosomes There is a marked (at least 10-fold) increase in antibody-dependent cellular cytotoxicity (ADCC) against both erythroid and tumor targets accompanied by increased expression of Fc receptors (FcR) There is also an augmentation of antibody-dependent phagocytosis Medium conditioned by unstimulated lymphocytes, a B lymphoblastoid cell line or a fibroblast cell line produce very little stimulation of U937 cells U937 cells gradually lose their increased ADCC capacity if they are washed free of conditioned medium (CM) but can be maintained in a stimulated state for long periods of time by culturing them in medium supplemented with PHA-CM at a concentration of 2% Stimulation of macrophage-like cell line U937 may provide a useful model for further study of mechanisms of macrophage cytotoxicity and its modulation by products of activated lymphocytes

Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies.

Abstract: This study on human neutrophils was conducted to measure the kinetics of degranulation of the different cytoplasmic granules into phagocytic vacuoles, and to relate the timing of these events to the burst of respiration that accompanies phagocytosis by these cells. Purified neutrophils were incubated with latex particles opsonized with human immunoglobulin (Ig)G, and phagocytosis was stopped at timed intervals. The cells were examined by electron microscopy to document the sequence of degranulation of the cytoplasmic granules. The azurophil granules and lyosomes were identified by histochemical staining for peroxidase and acid phosphatase, respectively. Phagocytic vacuoles were separated from cell homogenates by floatation on sucrose gradients and assayed for contained lactoferrin, myeloperoxidase, and acid hydrolases. The conclusions drawn from the biochemical and morphological studies were in agreement and indicated: particle uptake and vacuole closure can be completed within 20 s; both the specific and azurophil granules fuse with the phagocytic vacuole much earlier than is generally appreciated, with half-saturation times of 39 s (99% confidence limits, 15-72); oxygen consumption has kinetics similar to those of the fusion of these granules with the phagosome; degranulation of the acid hydrolases beta-glucuronidase, N-acetyl-beta-glucosaminidase (biochemical assays), and acid phosphatase (biochemical assay and electron microscopic cytochemistry) have kinetics of degranulation that are similar to each other but totally different from and much slower than that of myeloperoxidase with half-saturation times of between 354 and 682 s (99% confidence limits, 246-883). This suggests that the acid hydrolases are not co-located with myeloperoxidase in the azurophil granule but are contained in distinct lysosomes, or "tertiary granules".

Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages

Abstract: Several approaches have been used to study the determinants of phagosome-lysosome fusion in intact mouse macrophages. Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy. Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes. Good agreemen samples with an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology. The rate of usion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or nondigestible substances. With this assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate. Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found by use of the EM assays. Fusion was strongly affected by incubation temperature, having a Q10 of 2.5 No detectable fusion occurred below 15 degrees C, and this inhibition was rapidly reversed when cells were returned to 37 degrees C.

The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination.

Abstract: A method has been developed to deliver an idoinating system into the confines of the phagolysosome, allowing us to study the nature of the phagolysosomal membrane. Lactoperoxidase (LPO) is covalently coupled to carboxylated latex spheres (LPO-latex) in a stable, enzymatically active form. The addition of LPO-latex to cultured macrophages leads to their rapid attachment, ingestion, and enclosure in a plasma membrane-derived phagocytic vacuole. These organelles rapidly fuse with preexisting lysosomes and are converted to phagolysosomes (PL) that demonstrates both acid phosphatase and lactoperoxidase activities. The exposure of LPO-latex containing cells to 125I- and an extracellular peroxide-generating system, glucose oxidase-glucose, at 4 degrees C leads to incorporation of label into TCA-precipitable material. The incorporated cel-associated label was present as monoiodotyrosine, and negligible amounts were found in lipids. Cell viability remained > 99%. Autoradiography at both the light and EM level revealed that > 97% of the cells were labeled, and quantitative analysis demonstrated the localization of grains to LPO-latex containing PL. PL were separated on sucrose gradients, and their radiolabel was confined almost exclusively to the membrane rather than soluble contents. SDS-polyacrylamide gel electrophoretic analysis of the peptides iodinated from within PL demonstrated at least 24 species with molecular weights ranging from 12,000 to 250,000. A very similar group of proteins was identified on the plasma membrane (PM) after surface iodination, and on latex phagosomes derived from iodinated PM. No novel proteins were detected in PL, either immediately after phagosome-lysosome fusion or after 1 h of intracytoplasmic residence. We conclude that the membrane proteins accessible to LPO-catalyzed iodination on the luminal surface of the PL and on the external face of the PM are similar, if not identical.

Alveolar Macrophage Ingestion and Phagosome-Lysosome Fusion Defect Associated with Virus Pneumonia

Abstract: Virus-induced suppression of pulmonary phagocytic defenses is associated with defects in the intracellular processing of bacteria by alveolar macrophages. To determine whether the intracellular defect is related to a failure in phagosomelysosome fusion, mice were infected with a sublethal dose of Sendai virus, and the capacity of phagocytic cells, obtained by lung lavage, to exhibit phagosomelysosome fusion was quantitated during the course of the viral infection. Lysosomes of alveolar macrophages were prelabeled with acridine orange, the cells were challenged with Candida krusei, and fusion was determined with fluorescence microscopy by the discharge of the dye into the yeast-containing phagosome. Ultrastructural cytochemical studies verified the validity of the fluorescent fusion assay. Simultaneous experiments were performed to determine whether the viral infection also suppressed phagocytic ingestion by alveolar macrophages. Phagosome-lysosome fusion was progressively inhibited during the viral infection, reaching a low at day 7 when only 13 ± 3% of the phagocytic cells fused as compared with 97 ± 3% in cells from uninfected control animals; respectively, 55 ± 5% as compared with 74 ± 2% of the phagocytic cells contained yeasts. Thereafter, phagosome-lysosome fusion progressively increased reaching near normal levels (92 ± 3%) on day 17 of the infection. At the same time period, phagocytic uptake was enhanced to a level where 97 ± 3% of the cells contained yeasts. These data demonstrated that virus-induced suppression of intrapulmonary killing of bacteria involves functional lesions that retard the ingestion of inhaled organisms by alveolar macrophages and inhibit intracellular processing by degradative lysosomal enzymes by interfering with phagosome-lysosome fusion.

The development of Trypanosoma cruzi in macrophages in vitro. Interaction with lysosomes and host cell fate.

Abstract: The interaction between mouse peritoneal macrophages and ‘Y’ strain Trypanosoma cruzi bloodstream forms was studied at optical and electron microscopical levels. The method of marking lysosomes with Thorotrast, either before or after infection of cell monolayers with parasites, revealed that secondary lysosomes fused with phagosomes shortly after trypanosome interiorization. In spite of this, 24 h later most parasites were no longer in a vacuole but lay free within the host cell cytoplasm, multiplying actively. At this time, and up to shortly before 96 h when parasites escaped to the external milieu, most parasitized cells were not lethally injured, as revealed by the Trypan blue dye-exclusion test. Only when parasites were released into the external medium was this situation reversed and infected macrophages took up the dye.

A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba.

Abstract: Particle ingestion by Acanthamoeba is rapid. Within 40 s bound particles can be surrounded by pseudopods, brought into the cytoplasm, and released as phagosomes into the cytoplasmic stream. In electron micrographs the phagosome appears as a flasklike invagination of the surface. Separation from the surface occurs by fragmentation of the attenuated "neck+ of the invagination. The separated phagosome membrane has a three- to fourfold greater density of intramembrane particles than the plasma membrane from which it derives. This change is evident within 15 min of ingestion and is detectable while the membrane is still tightly apposed to the particle. There is no direct evidence for the mechanism of this increase; no increase in particle density was seen in the membrane at an early stage in the forming phagosomes still connected to the surface. These morphological observations are consistent with chemical analyses, to be reported in a separate communication, that show that the phagosome membrane has a higher protein to phospholipid ratio and a higher glycosphingolipid content than the plasma membrane. Enlarged phagosomes (presumptive phagolysosomes) show multiple small vesiculations of characteristic morphology. The small vesicles are postulated to be the major route of membrane return to the cell surface.

Electron-microscope study of Dictyostelium discoideum plasma membrane and its modifications during and after phagocytosis.

Abstract: The study of plasma membrane and phagosome membrane of Dictyostelium discoideum was performed using 2 lectins: concanavalin A (Con A) and Wheat Germ Agglutinin (WGA), and 2 markers of anionic sites: colloidal iron hydroxide (CIH) and cationized ferritin (CF). These labellings were applied to fixed partially broken cells, which had ingested a large quantity of yeast. They showed that Con A and CF labelled both the outer and inner faces of the plasma membrane, whereas CIH and WGA were deposited on the outer face only. Phagosome membranes displayed the same location as the plasma membrane for Con A, CF and CIH even in very old phagosomes. This suggests that most receptors of these 3 markers were not degraded by hydrolases. In contrast, phagosome membranes of young and old phagosomes did not react with WGA. When labellings were made on yeast phagocytozing cells, the membrane of phagocytic cups were also devoid of WGA, while it was labelled with the 3 other markers. The absence of WGA labelling was not observed during ingestion of bacteria and latex beads, suggesting a specific relationship existed between yeast cells and WGA receptors.

Interaction of Nocardia asteroides with rabbit alveolar macrophages: effect of growth phase and viability on phagosome-lysosome fusion.

Abstract: Rabbit alveolar macrophages were infected in vitro with cells of Nocardia asteroides GUH-2 in either logarithmic or early or late stationary phases of growth. Previous studies have established that during the growth cycle dramatic changes occur both in cell wall composition and structure and in the virulence of this organism. This study establishes the correlation between the relative virulence of the phase of growth of the infecting organisms and the degree of inhibition of macrophage phagosome-lysosome fusion. The occurrence of phagosome-lysosome fusion in infected macrophages was determined by both fluorescent and electron microscopy. It was found that relatively few phagosomes containing the highly virulent log-phase organisms had any evidence of lysosomal fusion; more of the phagosomes containing early stationary-phase cells had evidence of fusion. The greatest amount of phagosome-lysosome fusion was observed with the least virulent late stationary-phase cells. Electron microscopic evaluation of infected macrophages indicated that this increase in fusion was not associated with an increase in cell damage. Comparison of macrophages infected with either viable or nonviable organisms indicated that loss of viability did not decrease inhibition of fusion by early or late stationary-phase cells. In contrast, loss of viability did decrease inhibition of fusion by log-phase cells.

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils.

Abstract: Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.

Factors Modifying the Fusion of Phagosomes and Lysosomes: Art, Fact, and Artefact

Abstract: In their heuristic studies, Fell and Mellanby (1950,1952) described the destructive effects of vitamin A acetate on limb-bone rudiments and other tissues from mouse and chick embryos in tissue culture. The dissolution of, e.g., cartilage muco-protein associated with the tissue destruction was ultimately linked to proteolytic (cathepsin) activity released from tissue lysosomes which were rendered fragile and unstable, probably owing to a drug-induced labilization of lysosomal (and other) membranes.

Determinants of Phagosome-Lysosome Fusion in Mouse Macrophages

Abstract: Membrane fusion is an important event in many cell activities. It occurs between cells themselves, as in fertiliziation, myo-blast fusion, and virus or polyethylene glycol-induced cell-cell fusion. Some viruses fuse with cells to infect them, and many bud from the cell membrane during replication, requiring fusion to complete their maturation. Membranes fuse to close endocytic vesicles of various sizes and to separate two daughter cells during cytokinesis. Secreting cells fuse exocytic vesicles with their plasma membrane. Phagosomes fuse with lysosomes to begin the process of intracellular digestion. Little is known about these physiological fusion processes. Most work has been done in several model systems, which perhaps give some hints about possible molecular mechanisms.

Manipulations of Phagosome-Lysosome Fusion in Cultured Macrophages: Potentialities and Limitations

Abstract: Some pathogens, prominently Mycobacterium tuberculosis (Armstrong & Hart 1971; Draper & Hart 1975) and Toxoplasma gondii (Jones & Hirsch 1972; Jones 1975), do not induce the fusion of lysosomes with phagosomes that usually occurs after ingestion of foreign bodies, including many micro-organisms, by cultured macrophages. These and subsequent observations have provided some new insight into intracellular survival.

Phagocytosis in polycelis tenuis

Abstract: On feeding, occlusion of the intestinal lumen has been observed in this organism. Columnar phagocytes from opposing sides come together and interdigitate apically, forcing food particles, consisting of semi-digested tissues and cells, into the gastrodermis. In some instances, the phagocytosis of intact cells can be seen. Using horse-radish peroxidase as a marker, phagocytosed material can be histochemically detected a few minutes after commencement of feeding. Mixing of phagosomes and lysosomes occurs between 30 minutes and 2 hours after feeding, and digestion continues for up to 5 hours, as demonstrated by the presence of acid phosphatase. The fine structure of the gastrodermis is reviewed and the relationships between phagocytosis, enzyme production and intracellular digestion investigated. Primary lysosomes containing acid phosphatase appear to be formed at the Golgi apparatus, basally in the gastrodermal phagocytes, and then fuse with the incoming phagosomes, forming digestive vacuoles or secondary lysosomes. The digestive vacuoles finally end up as residual bodies. Autophagy and cell death are demonstrated and their role in nutrition is discussed.

Vital fluorescence microscopy of lysosomes in cultured mouse peritoneal macrophages during their interactions with microorganisms and active substances. III. Interactions of macrophages with endozoits of Toxoplasma gondii RH strain and their soluble substance.

Abstract: Macrophages with lysosomes pinpointed by quinacrine-induced fluorescence were infected with the endozoits of Toxoplasma gondii RH strain (peritoneal exudate of infected mouse), or treated with liquid (acellular) fraction of the same exudate. Dead toxoplasmas ingested by macrophages come into contact with the stained lysosomes of the cell and acquire a diffuse fluorescence. Viable toxoplasmas do not give fluorescence, which means that they do not come into contact with lysosomes, either primary or secondary. This supports the hypothesis that toxoplasmas can prevent lysosomes from fusing with the phagosomes of the host cell. Moderate doses of soluble products of toxoplasmas contained in peritoneal exudate cause an excessive output of macrophage lysosomes which points to the activation of macrophages; high doses of challenge inhibit the phagocytosis of toxoplasmas and damage macrophages. The pathogenicity of toxoplasmas due to their ability to inhibit the fusion of lysosomes and phagosomes and the cellular action of their soluble products is discussed.

The role of leukocyte extracts and myeloperoxidase in the lysis of staphylococci and the inhibition of bacteriolysis by anionic polyelectrolytes and by inflammatory exudates.

Abstract: Although much is known today about the mechanisms by which leukocytes and their intracellular enzymes kill bacteria very little is known about the biochemical pathways of degradation of the ingested microbiota. It is accepted that soon after the uptake of bacteria by neutrophils (PMNs) and macrophages fusion of lysosomes with the phagosome occurs. This is followed by the intravacuolar discharge of lysosomal agents which lead to the killing and degradation of the intracellular bacteria (2).