Showing papers on "Phagosome published in 1986"

Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium.

Abstract: Bone marrow-derived cultured macrophages were infected with the pathogenic organism Mycobacterium avium. Immediately after infection and at 1 to 28 days later, cells either were stained for acid phosphatase activity or given horseradish peroxidase, which served as a pinocytotic marker. With the former, fusions between phagosomes and lysosomes exclusively were assessed; with the latter, those between phagosomes and both pinosomes and lysosomes were determined. As a control, similar experiments were undertaken by infecting macrophages with gamma ray-killed M. avium and the nonpathogenic live organisms Mycobacterium aurum and Bacillus subtilis. After infection with live M. avium, fusions between phagosomes and acid phosphatase-positive vesicles (lysosomes) were inhibited. The same inhibition was observed whether phagosomes contained damaged or structurally intact (presumed to be live) bacteria, except for the early time points. This inhibition was, however, partial, suggesting that some of the live bacteria are resistant to the hydrolytic enzymes of the phagolysosomal environment. Fusions between horseradish peroxidase-positive vesicles (pinosomes and lysosomes) and phagosomes depended upon the morphological state of the bacteria. Damaged bacteria did not inhibit fusions, whereas with intact bacteria, a partial inhibition which increased with time was observed. The two types of experiment suggest that viable M. avium can impair phagosome-pinosome fusions.

Phagocytosis of Staphylococcus aureus by cultured bovine aortic endothelial cells: model for postadherence events in endovascular infections.

Abstract: We examined the interaction of Staphylococcus aureus with cultured bovine aortic endothelial cells as a model for the initial events in the pathogenesis of endovascular infections. Confluent monolayers of cultured endothelial cells were incubated with S. aureus. Cell-associated bacteria were measured by washing away nonadherent organisms, disrupting the monolayers, and performing quantitative cultures. Phagocytosis was differentiated from adherence by treating the cells with lysostaphin; approximately 60% of cell-associated bacteria was found to be intracellular. Phagocytosis could be blocked by using cytochalasin B, which interferes with microfilament function. Addition of fibronectin resulted in a 63% increase in adherence of S. aureus to the endothelial cells but did not increase ingestion. Transmission electron microscopy demonstrated a sequence of events similar to that which occurs during ingestion by professional phagocytes, including: adherence of bacteria to the endothelial cell; formation and elongation of surface extensions of the endothelial cell to surround the adherent bacteria; and complete enclosure within apparent phagosomes. Phagocytosis of bacteria by endothelial cells, followed by intracellular persistence, may be an important postadherence event in the pathogenesis and pathophysiology of endovascular infections.

Toxoplasma Modifies Macrophage Phagosomes by Secretion of a Vesicular Network Rich in Surface Proteins

Abstract: Modification of macrophage phagosomes begins shortly after formation as Toxoplasma cells secrete membranous vesicles that form a reticulate network within the vacuole. The Toxoplasma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein. The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxoplasma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular Toxoplasma cells as shown by immunoperoxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles. Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the Toxoplasma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.

Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila.

Abstract: Legionella pneumophila multiplied rapidly in guinea pig and rat alveolar macrophages but failed to grow when phagocytic activity was inhibited by pretreatment with 0.5 or 1.0 microgram of cytochalasin D per ml. Attachment was not inhibited by cytochalasin D. No extracellular multiplication occurred when L. pneumophila were in close proximity to viable functional macrophages or even when the bacteria were attached to plasma membranes of the macrophages. Nonopsonized L. pneumophila were avidly phagocytized by alveolar macrophages. When bacteria were centrifuged onto a cell pellet, more than 85% of the phagocytes contained one or more bacteria within 15 min. In contrast, under the same conditions only approximately 15% of the macrophages contained nonopsonized Escherichia coli or Staphylococcus aureus. Phagocytosis of L. pneumophila by untreated guinea pig macrophages occurred by extension of pseudopodia around the bacteria in a classical manner. The failure of the bacteria to actively penetrate the phagocyte suggests that their intracellular survival must not depend on avoidance of a phagosome but rather on an inhibition of or resistance to subsequent microbicidal functions of the macrophage. Images

Fate of Chlamydia trachomatis in human monocytes and monocyte-derived macrophages.

Abstract: The fate of Chlamydia trachomatis (L2/434/Bu) in human peripheral blood monocytes and human monocyte-derived macrophages was studied by transmission electron microscopy (TEM) and by measuring the yield of infectious C. trachomatis in one-step growth experiments. Two main types of phagosome were seen by TEM in the cytoplasm of C. trachomatis-infected human monocytes (1 h postinfection [p.i.]): one in which the elementary body (EB) was tightly surrounded by the membrane of the phagosome and another in which the EB appeared in an enlarged phagosome. Later, 24 to 48 h p.i., each phagosome contained a single EB-like particle, an atypical reticulate body, or a damaged particle. One-step growth experiments showed that infection of human monocytes with C. trachomatis results in a decrease of infectious particles between 24 and 96 h p.i., whereas infection of the monocytes by C. psittaci (6BC strain) results in productive infection with, however, a 3.5-log lower yield than in control MA-104 cells. In contrast to the abortive replication of C. trachomatis in monocytes, monocyte-derived macrophages permitted replication as indicated by one-step growth experiments and TEM. in C. trachomatis-infected, monocyte-derived macrophages 72 h p.i., inclusions of two kinds were observed by TEM. One was very similar to the typical inclusions appearing in infected MA-104 (control) cells; the other was atypical, pleomorphic, often contained "channels," and held relatively few EB and reticulate bodies, some of which appeared damaged or abnormal. The significance of the responses to infection with C. trachomatis in monocytes compared with monocyte-derived macrophages and the role of these cells in sustaining chronic or latent infection and in dissemination of the infection to various parts of the body is discussed.

Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro.

Abstract: Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues.

Phagosome-lysosome fusion

Abstract: Phagosome-lysosome fusion plays an important role in the destruction of micro-organisms by phagocytic cells. Several approaches have been used to study the determinants of phagosome-lysosome fusion in mouse macrophages. Fusion can be assayed by electron microscopy using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes or by a rapid and quantitative fluorescence assay. In the second method, secondary lysosomes are labelled with the fluorescent vital dye Acridine Orange and the rate and extent of their fusion with yeast-containing phagosomes is monitored by fluorescence microscopy (Hart & Young, 1975; Kielian & Cohn, 1980). Good agreement was found with the results obtained from both methods. The rate of fusion is not affected by the number of particles ingested, by enzymic modification or by concanavalin A cross-linking of the plasma membrane or by coating the phagocytic particle with concanavalin A or immune serum (Kielian & Cohn, 1980, 1981~). Fusion is also independent of drugs that affect the cytoskeleton, such as colchicine and cytochalasin B (Kielian & Cohn, 198 la). A limited number of factors can modulate the fusion process either positively or negatively. The rate and extent of fusion is dramatically increased after several days of cell culture. In 4-day cells, the initial rate of fusion is about eightfold higher than at 5 h (Kielian & Cohn, 1980). Fusion is also highly increased in macrophages activated in vivo or treated with the tumour promoter phorbol myristate acetate (PMA) at concentrations of 0.1-1 .Opg/ml (Kielian & Cohn, 1981b). Macrophages require 2-3 h of pretreatment with PMA to express maximal phagosome-lysosome fusion, and the effect of PMA stimulation is maintained for at least 20 h when cells are returned to PMA-free medium. The protein synthesis inhibitors puromycin and cycloheximide block the enhancement of phagosome-lysosome fusion, suggesting that PMA-stimulated phagosomelysososme fusion requires protein synthesis. The high fusion rate of 4-day cells is not increased by PMA and is unaffected by protein synthesis inhibitors. Fusion is inhibited by decreased temperature (Kielian & Cohn, 1980). No detectable fusion occurs below 15°C and this inhibition is rapidly reversed when cells are returned to 37°C. Fusion is also inhibited by lysosomal uptake of a number of polyanionic compounds containing high densities of sulphate, sulphonate or carboxylate residues (Hart & Young, 1980). Dextran sulphate (DS) in pg/ml quantities is an excellent inhibitor, whereas nonsulphated dextran is without effect at 1000-fold higher concentrations (Kielian et al., 1982). The DS inhibition correlates with its accumulation in high concentration

Respiratory burst during phagocytosis: an overview.

Abstract: Publisher Summary This chapter provides an overview of the respiratory burst during phagocytosis. Granulocytes, some classes of macrophages, and natural killer cells release substantial amounts of superoxide (O 2 – ) and hydrogen peroxide (H 2 O 2 ) when their plasmalemma is perturbed. This response and the concomitant phenomena is termed as the “respiratory burst.” The perturbations induced by physiological processes can be mimicked by certain soluble or “quasi-soluble” agents. Moreover, superoxide and H 2 O 2 , formed on the external surface of the plasmalemma and internal surface of the phagosome, may interact to form hydroxyl radical and perhaps singlet oxygen. The most thoroughly studied cidal system known to function in the phagosome consists of myeloperoxidase, H 2 O 2 , and chloride. This system has been shown in vitro to be effective in killing bacteria, yeast, mycoplasma, viruses, and tumor cells. The complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity consists of at least two components—namely, a flavoprotein and a b -cytochrome with a low midpoint potential.

[11] Assay of phagosome-lysosome fusion

Abstract: Publisher Summary This chapter discusses the assay of phagosome–lysosome (P–L) fusion. Particles ingested by cells are contained within a membrane-bounded vacuole or phagosome. In many cases, this vacuole undergoes fusion with lysosomes, resulting in exposure of the particle to both the acidic pH and hydrolytic enzymes of the lysosome. Electron microscopy has been used to look for the appearance of lysosomal markers, such as acid phosphatase or exogenously fed peroxidase, ferritin, colloidal gold, or thorium dioxide in the phagocytic vacuole. Lysosomes are labeled with a fluorescent marker, and fluorescence microscopy is used to follow transfer of the marker into phagocytic vacuoles. This assay is simple, rapid, enables detailed and reproducible rate studies, and makes possible the analysis of P–L fusion using intact and viable cells as a test system. Cultured cells are prelabeled with acridine orange (AO), a fluorescent vial dye that by its weakly basic nature becomes concentrated primarily in the low pH environment of lysosomes. Phagocytosis is essentially complete by the first time point of the fusion assay, and the rate of subsequent P–L fusion may be followed independent of the phagocytic rate.