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- Majeed, Meytham, et al.
(författare)
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Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils
- 1998
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Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 24:5, s. 309-320
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Tidskriftsartikel (refereegranskat)abstract
- The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]iand certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.
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- Serrander, Lena
(författare)
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Neutrophil phagocytosis and secretion : The role of calcium and the cytoskeleton
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neutrophils assure rapid removal of bacteria by a variety of processes. They crawl out of the vessels, phagocytose the bacteria and kill them by secretion of bactericidal substances and production of oxidative metabolites. The aims of this study were to investigate the signalling pathways during these processes, in particular i) how complement receptors mediate phagocytosis and NADPH~oxidase activity ii) the role of Ca2+ in secretion and the role of a Ca2+-dependent, actin-binding protein, _gelsolin, in neutrophil phagocytosis and secretion. Conventional biochemical methods, capacitance measurements of secretion with the patchclamp technique and visualisation with fluorescence microscopy techniques were used. We found that phospholipase D (PLD) is an early Ca2+-independent signal in complementmediated phagocytosis, preceding cytoskeletal rearrangements. We also demonstrated'that the NADPH-oxidase could be activated in situ to generate oxidative metabolites intracellularly after particle stimulation of complement receptors in the absence of phagocytosis. This permits cells to use oxidative metabolites for signalling and not only to kill bacteria. This activation involved the cytoskeleton and PLD. Whereas signalling during pha~ocytosis can occur independently of Ca2+, other neutrophil functions are highly Ca +-dependent. Investigating the relative importance of Ca2+-release from intracellular stores versus Ca2+ influx over the plasma membrane, we found that secretion of primary granules induced by fMLP is dependent on Ca2+ -influx. ci+-influx alone is not sufficient to induce secretion in neutrophils. A second synergistic signal is required. This missing signal was not PLD, PLC or tyrosine kinases, but involved a pertussis-sensitive 0-protein and PI3-kinase. When further investigating the role of Ca2+ in secretion, we found that secretion of different granules is regulated by different [Ca2+:J. Primary granules are secreted at 100 ~ Ca2+ whereas the other granules are secreted at 1.5-5 ~ ci+, suggesting two mechanisms involving different Ca2+ activated systems/sensor proteins. One sensor protein could be the Ca2+-dependent, actinbinding protein, gelsolin, which has earlier been shown to stimulate secretion in different celltypes. Secretion from gelsolin-deficient mouse neutrophils was nevertheless nonnal. Gelsolin was however found to be essential for !gO-mediated-, but not complement-induced phagocytosis. Activation of the oxidase and phagolysosorne-fusion was unaffected in gelsolin-deficient neutrophils. This suggests gelsolin to be a Ca2+ -sensor specifically for !gOmediated phagocytosis. !gO-mediated phagocytosis often leads to more efficient killing than complement-mediated phagocytosis. Gelsolin seems to be part of the machinery that distinguishes the two pathways of phagocytosis. The present data show that receptor mediated activation of neutrophil functions involves several signalling pathways. This allows selective modulation of the inflammatory response.
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- Zimmerli, Stefan, et al.
(författare)
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Phagosome-Lysosome Fusion Is a Calcium-independent Event in Macrophages
- 1996
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 132:1-2, s. 49-61
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Tidskriftsartikel (refereegranskat)abstract
- Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms, Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+](i)). Indeed, increases in [Ca2+](i) are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes, Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (MO) were treated with 12.5 mu M bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to less than or equal to 20 nM and completely blocked increases in [Ca2+](i) in response to multiple stimuli, even in the presence of extracellular calcium, Subsequently, MO phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis, Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes, Confirmation of phagosomelysosome fusion by electron microscopy validated the fluorescence microscopy findings, We found that phagosome-lysosome fusion in MO occurs normally at very low [Ca2+](i) (less than or equal to 20 nM), Kinetic analysis showed that in MO none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+](i) in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs, control macrophages, We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.
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