scholarly journals Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival.

1975 ◽  
Vol 142 (1) ◽  
pp. 1-16 ◽  
Author(s):  
J A Armstrong ◽  
P D Hart
Keyword(s):  
Electron Microscopy ◽  
Rabbit Antiserum ◽  
Peritoneal Macrophages ◽  
Bacterial Survival ◽  
Virulent Challenge ◽  
Secondary Lysosomes ◽  
High Level ◽  
Mouse Peritoneal Macrophages ◽  
Specific Rabbit ◽  
Strain H37rv

Tubercle bacilli of the pathogenic human strain H37Rv had previously been shown to multiply, after ingestion by cultured mouse peritoneal macrophages, within phagosomes that tended to remain unfused with secondary lysosomes. Means were sought therefore for promoting experimentally a modification of the host response so as to attain a high level of phagolysosome formation, enabling tests to be made of any effects on the course and outcome of the intracellular infection. This was achieved by exposing viable bacilli to specific rabbit antiserum before their ingestion. Quantitative assessments, using electron microscopy, now showed that a majority of the phagosomes containing intact bacilli had fused with ferritin-labeled lysosomes, and frequently the fusion was massive. Bacterial viability studies established that the serum pretreatment was not itsel bactericidal. In the course of progressive infections with strain H37Rv, monitored by counts both of viable bacterial units and of intracellular acid-fast organisms, no appreciable difference was found between the intracellular growth rates of control and antiserum-treated bacilli. Concurrent electron microscopy showed that bacilli could remain intact and multiply both in phaagolysosomes and in unfused phagosomes, ruling out the possibility of selective growth of antiserum-pretreated bacilli within the minority of phagosomes that remained unfused. It was concluded that "turning on" phagosome-lysosome fusion in normal macrophages did not influence the outcome of infection with virulent M. tuberculosis; lysosome contents manifestly failed to exercise an antibacterial effect on this organism. Nevertheless, the possibility remains that the lysosomes of specific immune macrophages have antituberculous potentiality. In that case the experimental "turning on or off" of fusion could be a decisive factor in the outcome of a virulent challenge. Should it not be, the antibacterial capabilities of immune cells would need to be ascribed to factors other than lysosomal attack, the latter being essentially for disposal of the dead organisms.

Changes of the phagosomal elemental concentrations by Mycobacterium tuberculosis Mramp

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 323-332 ◽  
Author(s):  
Dirk Wagner ◽  
Jörg Maser ◽  
Ivana Moric ◽  
Neio Boechat ◽  
Stefan Vogt ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Divalent Cations ◽  
Peritoneal Macrophages ◽  
Bacterial Survival ◽  
Knockout Mutant ◽  
Elemental Concentrations ◽  
Restricted Environment ◽  
Mouse Peritoneal Macrophages ◽  
Strain H37rv

Pathogenic mycobacteria survive within phagosomes which are thought to represent a nutrient-restricted environment. Divalent cation transporters of the Nramp family in phagosomes and mycobacteria (Mramp) may compete for metals that are crucial for bacterial survival. The elemental concentrations in phagosomes of macrophages infected with wild-type Mycobacterium tuberculosis (M. tuberculosis strain H37Rv) and a M. tuberculosis Mramp knockout mutant (Mramp-KO), derived from a clinical isolate isogenic to the strain MT103, were compared. Time points of 1 and 24 h after infection of mouse peritoneal macrophages (bcg S) were compared in both cases. Increased concentrations of P, Ni and Zn and reduced Cl concentration in Mramp-KO after 1 h of infection were observed, compared to M. tuberculosis vacuoles. After 24 h of infection, significant differences in the P, Cl and Zn concentrations were still present. The Mramp-KO phagosome showed a significant increase of P, Ca, Mn, Fe and Zn concentrations between 1 and 24 h after infection, while the concentrations of K and Ni decreased. In the M. tuberculosis vacuole, the Fe concentration showed a similar increase, while the Cl concentration decreased. The fact that the concentration of several divalent cations increased in the Mramp-KO strain suggests that Mramp may have no impact on the import of these divalent cations into the mycobacterium, but may function as a cation efflux pump. The concordant increase of Fe concentrations within M. tuberculosis, as well as within the Mramp-KO vacuoles, implies that Mramp, in contrast to siderophores, might not be important for the attraction of Fe and its retention in phagosomes of unstimulated macrophages.

Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages

1990 ◽  
Vol 68 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Mayer B. Goren ◽  
Natan Mor
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Dextran Sulfate ◽  
Peritoneal Macrophages ◽  
Streptococcus Faecalis ◽  
Viable Bacteria ◽  
Secondary Lysosomes ◽  
Mycobacterium Lepraemurium ◽  
Mouse Peritoneal Macrophages

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome–lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believe it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-μm carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.Key words: phagosomes, lysosomes, fusion, polyanions, electron microscopy.

scholarly journals RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

1971 ◽  
Vol 134 (3) ◽  
pp. 713-740 ◽  
Author(s):  
J. A. Armstrong ◽  
P. D'Arcy Hart
Keyword(s):  
Early Stage ◽  
Virulent Strain ◽  
Peritoneal Macrophages ◽  
Thin Sections ◽  
Dense Granules ◽  
Direct Exposure ◽  
Significant Difference ◽  
Steady Level ◽  
Secondary Lysosomes ◽  
Strain H37rv

The cytological response to the ingestion of tubercle bacilli by cultured mouse peritoneal macrophages has been studied by electron microscopy. Methods included a quantitative assessment based on systematic surveying of cell profiles, and of phagosomes and their contained bacteria, encountered in thin sections; classification of the sectioned bacteria into visibly damaged and apparently intact categories; prelabeling of dense granules (secondary lysosomes) with ferritin as an aid to identifying the occurrence and frequency of phagosome-lysosome fusion; and monitoring of bacterial growth and viability by light microscopy and cultural counts. The situations studied were as follows: progressive infection with the multiplying virulent strain H37Rv; ingestion of the same strain previously inactivated by gamma radiation; infection with an attenuated strain (BCG); and a stabilized virulent infection induced by the surfactant Macrocyclon. In the bacterial suspensions used routinely for inoculation, about half the bacilli were viable, matching closely the proportions of intact and damaged organisms identified with the electron microscope. In the inoculated macrophages, some phagosomes containing intact bacilli and others containing damaged bacilli were always to be found; but the proportion of organisms scored as damaged increased, and that of intact organisms decreased, in situations where the population as a whole had been rendered nonviable before inoculation, or where they became so intracellularly as in the late stages of a BCG infection. Evidence of fusion of ferritin-marked lysosomes with some bacterium-containing phagosomes was obtained in all experiments, but a significant difference was regularly observed according to whether the bacilli were damaged or intact. Virtually all phagosomes containing damaged bacilli showed signs of fusion; but when many phagosomes were present containing apparently intact organisms (as with actively multiplying strain H37Rv or with this strain held at a steady level of viability by Macrocyclon, and also with strain BCG at an early stage of that infection), signs of fusion of lysosomes with these phagosomes were infrequent. From these findings it is inferred that intracellular survival of M. tuberculosis in cultured macrophages is associated with a tendency to nonfusion of dense granules with the phagosome, thus avoiding direct exposure of the bacilli to the contents of these organelles. It is suggested, further, that fusion of dense granules with the phagosome, leading to digestion, is determined by recognition of the bacillus as nonviable. The possibility is discussed that the cytological response to different mycobacterial infections may reflect differences of a basic nature between facultative and obligate intracellular parasitism.

scholarly journals EFFECTS OF CONCANAVALIN A ON MOUSE PERITONEAL MACROPHAGES

1974 ◽  
Vol 140 (5) ◽  
pp. 1364-1386 ◽  
Author(s):  
Paul J. Edelson ◽  
Zanvil A. Cohn
Keyword(s):  
Concanavalin A ◽  
Peritoneal Macrophages ◽  
Surface Marker ◽  
Marker Enzyme ◽  
Apparent Effect ◽  
Con A ◽  
Cytoplasmic Vesicles ◽  
Secondary Lysosomes ◽  
Mouse Peritoneal Macrophages ◽  
Pinocytic Vesicles

Concanavalin A (Con A) binds to saccharide residues on the mouse peritoneal macrophage plasma membrane and stimulates extensive pinocytic interiorization of the membrane. The overall pinocytic rate is increased 3.5–4.5 times by the addition of Con A, and the surface marker enzyme adenosine triphosphatase can be identified histochemically in association with the cytoplasmic vesicles generated after exposure of the cells to Con A. Once formed, these pinocytic vesicles may persist for several days and fail to show morphologic evidence of fusion with primary or preformed secondary lysosomes. There is no apparent effect on the capacity of the macrophage to ingest either latex particles or IgG-coated SRBC administered either simultaneously with or subsequent to the Con A.

scholarly journals THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

1972 ◽  
Vol 55 (1) ◽  
pp. 186-204 ◽  
Author(s):  
Ralph M. Steinman ◽  
Zanvil A. Cohn
Keyword(s):  
Horseradish Peroxidase ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Wide Range ◽  
Mouse Macrophages ◽  
Secondary Lysosomes ◽  
Endocytic Vesicles ◽  
In Vitro Interaction ◽  
Mouse Peritoneal Macrophages

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 106 cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-125I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-125I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.

scholarly journals Interactions of TRIC agents with macrophages and BHK-21 cells observed by electron microscopy

1973 ◽  
Vol 71 (3) ◽  
pp. 515-528 ◽  
Author(s):  
A. M. Lawn ◽  
W. A. Blyth ◽  
Janice Taverne
Keyword(s):  
Electron Microscopy ◽  
Toxic Effect ◽  
Early Stage ◽  
Morphological Changes ◽  
Peritoneal Macrophages ◽  
Stage Of Development ◽  
Mouse Peritoneal Macrophages

SUMMARYTRIC agents do not multiply in mouse peritoneal macrophages in culture but have a toxic effect on them, whereas they multiply readily in BHK-21 cells. Sections of macrophages and of BHK-21 cells fixed during the first 90 min after inoculation were examined by electron microscopy. Macrophages ingested all forms of the organism, which were eventually degraded in lysosomes. However, elementary bodies were distinguished from other TRIC particles by the delay in their transfer to lysosomes. BHK-21 cells ingested elementary bodies selectively, and in these cells the organisms were neither found in lysosomes nor degraded. Instead they showed morphological changes that probably represented an early stage of development.

scholarly journals THE UPTAKE AND DIGESTION OF IODINATED HUMAN SERUM ALBUMIN BY MACROPHAGES IN VITRO

1967 ◽  
Vol 126 (5) ◽  
pp. 941-958 ◽  
Author(s):  
Barbara A. Ehrenreich ◽  
Zanvil A. Cohn
Keyword(s):  
Human Serum Albumin ◽  
Peritoneal Macrophages ◽  
Radioactive Material ◽  
In Vitro Cultivation ◽  
Cultivation Conditions ◽  
Secondary Lysosomes ◽  
Mouse Peritoneal Macrophages ◽  
Reduced Temperature ◽  
Pinocytic Vesicles

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 125I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of 125I-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 125I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.

scholarly journals Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis.

1983 ◽  
Vol 96 (3) ◽  
pp. 887-895 ◽  
Author(s):  
I S Mellman ◽  
H Plutner ◽  
R M Steinman ◽  
J C Unkeless ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Rabbit Antiserum ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Plasma Membrane Proteins ◽  
Coated Particles ◽  
Macrophage Receptors ◽  
Mouse Peritoneal Macrophages

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG-coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.

scholarly journals Inborn resistance of mice to myxoviruses: macrophages express phenotype in vitro.

1978 ◽  
Vol 147 (2) ◽  
pp. 531-540 ◽  
Author(s):  
J Lindenmann ◽  
E Deuel ◽  
S Fanconi ◽  
O Haller
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Peritoneal Macrophages ◽  
F1 Hybrids ◽  
Lethal Challenge ◽  
Avian Influenza A Virus ◽  
High Level ◽  
Mouse Peritoneal Macrophages

A strain of avian influenza A virus was adapted to grow in mouse peritoneal macrophages in vitro. The adapted strain, called M-TUR, induced a marked cytopathic effect in macrophages from susceptible mice. Mice homozygous (A2G) or heterozygous (F1 hybrids between A2G and several susceptible strains) for the gene Mx, shown previously to induce a high level of resistance towards lethal challenge by a number of myxoviruses in vivo, yielded peritoneal macrophages which were not affected by M-TUR. Peritoneal macrophages could be classified as resistant or susceptible to M-TUR without sacrificing the cell donor. Backcrosses were arranged between (A2G X A/J)F1 and A/J mice. 64 backcross animals could be tested individually both for resistance of their macrophages in vitro after challenge with M-TUR, and for resistance of the whole animal in vivo after challenge with NWS (a neurotropic variant of human influenza A virus). Macrophages from 36 backcross mice were classified as susceptible, and all of these mice died after challenge. Macrophages from 28 mice were classified as resistant, and 26 mice survived challenge. We conclude that resistance of macrophages and resistance of the whole animal are two facets of the same phenomenon.

scholarly journals Specialization of the macrophage plasma membrane at sites of interaction with opsonized erythrocytes.

1983 ◽  
Vol 96 (5) ◽  
pp. 1227-1233 ◽  
Author(s):  
R Montesano ◽  
A Mossaz ◽  
P Vassalli ◽  
L Orci
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Receptor Interaction ◽  
Coated Pits ◽  
Electron Dense Material ◽  
Functional Studies ◽  
Structural Specialization ◽  
Mouse Peritoneal Macrophages

We incubated mouse peritoneal macrophages for 3-8 min at 37 degrees C with antibody-coated sheep erythrocytes and examined regions of close interaction between the two cell types by electron microscopy. At sites of focal macrophage-erythrocyte contact we observed a distinctive specialization of the macrophage plasma membrane consisting of a prominent subplasmalemmal band of electron-dense material, approximately 25-35 nm in thickness. In many instances, this band showed a periodic substructure similar to that seen in clathrin coats. Moreover, many slender erythrocyte processes penetrated into invaginations of the macrophage surface which were bristle-coated at their blind extremity. As previously shown for clathrin-coated pits, the segments of the macrophage plasma membrane beneath which the defense material was found were selectively resistant to the membrane-perturbing effect of the antibiotic, filipin. This structural specialization of the macrophage plasma membrane at sites of ligand-receptor interaction during immune phagocytosis of antibody-coated erythrocytes may represent the morphological counterpart of the zipper mechanism of phagocytosis previously demonstrated by functional studies.

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