Showing papers on "Phagosome published in 1988"

Human Neutrophil Antimicrobial Activity

Abstract: Polymorphonuclear neutrophilic leukocytes (PMNs) take up opsonized microorganisms into phagosomes that fuse with secretory granules in the PMN cytoplasm to form phagolysosomes. Killing and digestion of microorganisms take place within phagolysosomes. Antimicrobial activities in phagolysosomes are divided into two classes. Oxygen (O2)-dependent mechanisms are expressed when PMNs undergo the "respiratory burst." An NADPH oxidase in the phagolysosome membrane is activated and reduces O2 to superoxide (O2-). O2 reduction is the first step in a series of reactions that produce toxic oxidants. For example, .O2- dismutases to hydrogen peroxide (H2O2), and the azurophil granule enzyme myeloperoxidase catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The reaction of HOCl with ammonia and amines modulates the toxicity of this oxidant. O2-independent antimicrobial mechanisms include the activities of lysosomal proteases, other hydrolytic enzymes, and proteins and peptides that bind to microorganisms and disrupt essential processes or structural components. For example, the bactericidal/permeability-increasing protein, cathepsin G, and the defensins are released into phagolysosomes from the azurophil granules. Proposed mechanisms of action of neutrophil antimicrobial agents, their range of microbial targets, and their possible interactions within phagolysosomes are discussed.

The effect of phenolic glycolipid-1 from Mycobacterium leprae on the antimicrobial activity of human macrophages.

Abstract: Purified PGL-1 and dPGL from M. leprae can prevent bacterial killing by intact phagocytes and cell-free antimicrobial systems. Both glycolipids completely abolished the antimicrobial effect of the acetaldehyde-XO-Fe2+ system. Because the cytotoxicity of this system is inhibited by catalase, SOD, mannitol, and ethanol, but not by heated SOD or catalase, these data suggest that toxicity is due to OH. generated by the Haber-Weiss reaction. That the antimicrobial killing in the XO system is completely blocked by the addition of PGL-1 or dPGL suggests that these glycolipids can act as OH. scavengers. A modest protective effect against the cytotoxicity of the MPO-H2O2-halide system by both PGL-1 and dPGL was also observed. The antimicrobial activity of the MPO system was abolished with chloride, but not iodide, as the halide. The effect of the M. leprae-derived glycolipid on bacterial killing by intact phagocytes was examined. Two linking antibodies were used to bind the dPGL to a rapidly growing test organism, S. aureus, a murine IgM mAb specific for the terminal glycoside of PGL-1, and a rabbit IgG anti-mouse IgM which bound the staphylococcal protein A via its Fc region. Examination by transmission EM of human monocyte-derived macrophages which had ingested staphylococci either coated with both antibodies and dPGL, or coated only with the IgG and IgM antibodies, demonstrated the presence of bacteria in phagosomes of control and IFN-gamma-activated macrophages. Activation of the macrophage monolayers by pretreatment with IFN-gamma markedly increased their staphylocidal activity. When dPGL coated staphylococci were ingested, killing by both control and IFN-gamma-activated macrophages was completely blocked. These results, suggesting that PGL-1 can scavenge reactive oxygen species and prevent microbial death within the phagosome, may in part explain the intracellular survival of M. leprae in certain cell types.

Phagosome-lysosome fusion in P388D1 macrophages infected with Histoplasma capsulatum.

Abstract: The issue of whether or not phagocytized Histoplasma capsulatum yeasts evade phagosome-lysosome fusion (P-LF) has been debated by several investigators. To resolve this problem, yet avoid drawbacks associated with the conventional assays of P-LF (electron microscopy and the acridine orange assay), we used fluorescein isothiocyanate-labeled dextran (FITC-dextran) to monitor P-LF in the macrophage-like cell line P388D1.D2. Controls indicated that FITC-dextran could be used to distinguish between evasion of P-LF by Toxoplasma gondii and phagolysosome formation following ingestion of Saccharomyces cerevisiae. Phagosomes containing H. capsulatum clearly fused with FITC-dextran-labeled lysosomes at a rate comparable to that observed for S. cerevisiae. This was true for several strains of H. capsulatum including two avirulent strains derived in this laboratory. Varying the dose of H. capsulatum did not alter the percentage of phagolysosomes formed. Our results indicate that H. capsulatum is one of a small number of organisms which is able to survive in phagolysosomes.

Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

Abstract: Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability.

Lysosomes and pancreatic islet function. A quantitative estimation of crinophagy in the mouse pancreatic B-cell.

Abstract: Ultrastructural studies of pancreatic islets have suggested that crinophagy provides a possible mechanism for intracellular degradation of insulin in the insulin-producing B-cells. In the present study, a quantitative estimation of crinophagy in mouse pancreatic islets was attempted by morphometric analysis of lysosomes containing immunoreactive insulin. Isolated islets were incubated in tissue culture for one week in 3.3, 5.5 or 28 mmol/l glucose. The lysosomes of the pancreatic B-cells were identified by morphological and enzyme-cytochemical criteria and divided into three subpopulations comprising primary lysosomes and insulin-positive or insulin-negative secondary lysosomes. Both the volume and numerical density of the primary lysosomes increased with increasing glucose concentration. The proportion of insulin-containing secondary lysosomes was highest at 5.5 and lowest at 3.3 mmol/l glucose. Insulin-negative secondary lysosomes predominated at 3.3 mmol/l glucose. Studies of the dose-response relationships of glucose-stimulated insulin biosynthesis and insulin secretion of the pancreatic islets showed that biosynthesis had an apparent Km-value for glucose of 7.0 mmol/l, whereas it was 14.5 mmol/l for secretion. The pronounced crinophagic activity at 5.5 mmol/l glucose may thus be explained by the difference in glucose sensitivity between insulin biosynthesis and secretion resulting in an intracellular accumulation of insulin-containing secretory granules. The predominance of insulin-negative secondary lysosomes at 3.3 mmol/l glucose may reflect an increased autophagy, whereas the predominance of primary lysosomes at 28 mmol/l glucose may reflect a generally low activity of intracellular degradative processes.

Invasiveness and intracellular growth of Listeria monocytogenes

Abstract: Listeria monocytogenes is an invasive bacterial pathogen capable of multiplying inside many host cells, including macrophages, enterocytes and hepatocytes. There is evidence to believe that secretion of listeriolysin O, an SH-activated exotoxin, is crucial for bacterial growth in host tissues. This exotoxin is stimulated in iron-deprived medium and mostly active at low pH (5.5). Electron microscopic studies showed that intracellular bacteria rapidly disrupt the vacuole membrane of phagosomes and freely multiply inside the cytosol of infected cells, thus escaping at an early stage of infection from the cellular microbicidal mechanisms. Vacuole disruption does not occur with a nonhemolytic mutant obtained by insertion of a single copy of transposon Tn1545 in the structural gene of listeriolysin O. These results strongly suggest that listeriolysin O is a major factor promoting intracellular growth ofL. monocytogenes and that intracellular growth of virulent bacteria is initiated after escaping from the phagosomal compartment.

Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils.

Abstract: To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.

Effects of pH on killing of Staphylococcus aureus and Escherichia coli by constituents of the neutrophil phagolysosome.

Abstract: Lysosomotropic weak bases impair in-vitro neutrophil functions including intracellular killing of Staphylococcus aureus strain 502a. To investigate whether prevention of phagosomal acidification could account for impaired microbicidal activity, a model phagosome was formulated with a freeze-thawed granule extract as a source of lysosomal enzymes and H2O2 as a source of toxic oxygen metabolites. The lysosomal extract alone killed Escherichia coli strain S15 efficiently at pH 5.5 and 7.0, but had little activity against S. aureus 502a. Sublethal concentrations of the two agents, when combined, acted synergically against either organism. Each organism was killed more effectively at pH 5.5 than at pH 7.0 by the lysosome extract-H2O2 combination, but the killing of E. coli was more rapid than that of S. aureus in the same conditions. These findings suggest that impairment of neutrophil antistaphylococcal activity by weak bases may be mediated by their ability to raise phagosomal pH, and that persistence of E. coli in similar conditions does not occur because the latter is killed by lysosomal constituents in a non-pH-dependent fashion.

Polyanionic agents inhibit phagosome-lysosome fusion in cultured macrophages: a reply to the suggestion of Goren, Vatter, and Fiscus to the contrary.

Abstract: The paper and review by Goren et al (J Leukocyte Biol 41, 111, 1987) contain serious objections to the reports from several laboratories on the pattern of fusion of secondary lysosomes with phagosomes (yeasts being predominantly the target) in polyanion-treated macrophages; these reports had concluded that the polyanions were inhibitors of this fusion The main objection by Goren et al is to the alleged misuse of electron microscopic (EM) lysosome markers; many instances of phagosome-lysosome (P-L) fusion in the treated cells have therefore been missed The central argument is that 1) the "hydrocolloid" properties of certain of these polyanions hinder the passage of the enmeshed marker from lysosome to phagosome after their fusion and 2) this hindrance is mistakenly interpreted as indicating that fusion has not taken place, thus giving rise to the belief that the polyanions can inhibit fusion In reply, we explain that we score as P-L fusion any instance of marker (ferritin) being seen anywhere in a fused phagosome (phagolysosome) For this crucial reason, immobilisation of marker by a hydrocolloid polyanion, eg, in lysosomal residue of phagolysosomes or just within phagosome membranes (as in Goren's Figs 5 and 6 [8]), would not seriously threaten the marker distinction between fusion and nonfusion (with consequent underestimation of the former) and therefore would not invalidate the reports of a high incidence of nonfusion in polyanion-treated macrophages Such inhibition of P-L fusion is supported by using as lysosomal label the nonpermeant fluorescent probe lucifer yellow (accepted by Goren et al as a reliable indicator of fusion) Further support comes from the correlated inhibition of the saltatory movements of secondary lysosomes previously described; static lysosomes will have their contact with the phagosomes severely restricted Another criticism is based on the failure of certain salient properties and functions of phagosomes to change significantly after polyanion treatment of the macrophages; these include intraphagosomal digestion, presence of lysosomal enzymes, and acidification However, this indirect evidence can be accounted for alternatively by the operation of factors (primary lysosomes, endogenous acidification, etc) not affected by the polyanionic block We conclude that fusion inhibition by the polyanions is a real phenomenon, as previously reported, notwithstanding the hydrocolloid properties of some of them Furthermore, an explanation based on hydrocolloid properties is questionable, since one or possibly two of the five main polyanionic agents appear not to be hydrocolloids

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils.

Abstract: An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.

The use of albumin-gold to follow lysosome-phagosome fusion.

Abstract: A new procedure for labeling of secondary lysosomes and to determine, by transmission electron microscopy, their fusion with phagosomes was developed. It is based on the use of albumin adsorbed to colloidal gold particles as a probe and tested using macrophages previously labeled with albumin-gold and then incubated in the presence of epimastigote forms of Trypanosoma cruzi.

Chapter 8 Polyanionic Agents and Inhibition of Phagosome-Lysosome Fusion: Paradox Lost

Abstract: Publisher Summary This chapter discusses polyanionic agents and the inhibition process of phagosome–lysosome fusion. It is concerned with a hypothesis about the antagonistic effects of endocytosed polyanionic substances on phagosome–lysosome fusion in cultured macrophages. It is observed that no important macrophage function has been discerned so far that is antagonized by lysosomal accumulation of polyanions—particularly of phagocytosis, intracellular digestion, and microbicidal activities. The apparent block to fusion is indifferent to Ca2+ concentrations and cannot be imposed from the phagosomal compartment by particles of cation-exchange resins. If this “phenomenon” depends on perturbations in vesicle membranes, pinosomal membranes would appear to be embarrassing exceptions to the inhibition. They will apparently behave like phagosomal membranes, both allowing fusion with “polyanion” lysosomes. Neutral hydrocolloids mimic the behavior of polyanions with thorotrast markers, but still allow delivery of permeant or nonpermeant mobile lysosomal fluors to phagosomes.

Effects of pH on killing of Staphy/ococcus aureus Escherichia cdiby constituents of the neutrophil p hagolysosome and

Abstract: Summary. Lysosomotropic weak bases impair in-vitro neutrophil functions including intracellular killing of Staphylococcus aureus strain 502a. To investigate whether prevention of phagosomal acidification could account for impaired microbicidal activity, a model phagosome was formulated with a freeze-thawed granule extract as a source of lysosomal enzymes and H202 as a source of toxic oxygen metabolites. The lysosomal extract alone killed Escherichia coli strain S 15 efficiently at pH 5.5 and 7.0, but had little activity against S. aureus 502a. Sublethal concentrations of the two agents, when combined, acted synergically against either organism. Each organism was killed more effectively at pH 5.5 than at pH 7.0 by the lysosome extract-H202 combination, but the killing of E. coli was more rapid than that of S. aureus in the same conditions. These findings suggest that impairment of neutrophil antistaphylococcal activity by weak bases may be mediated by their ability to raise phagosomal pH, and that persistence of E. coli in similar conditions does not occur because the latter is killed by lysosomal constituents in a non-pH-dependent fashion.