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Regulation of phagocytosis and phagolysosome fusion in human leukocytes
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Professional phagocytes such as neutrophil granulocytes and macrophages are an esential part of the innate immune system. The neutrophils form the first line of defence against invading microorganisms and are important for rapid killing of the intruders. Macrophages arrive later at the site of infection, kill micoorganisms, degrade dead cells, present antigens and secrete substances that orchestrate the inflammatory response. Neutrophils and macrophages ingest and kill microorganisms in a process called phagocytosis, where calcium signalling has shown to be involved. Inside the cell the microorganism is enclosed in a phagosome, that sequentially fuses with various intracellular vesicles to form a phagolysosome in which the intruder is killed. Killing is achieved through the actions of lytic enzymes, nitrogen oxide (NO) and reactive oxygen metabolites (ROM). Studies on the regulation of phagocytosis are essential since many pathogens are able to survive by interfering with this process. In the first study we investigated intracellular signalling in human neutrophils following engagement of a phagocytic receptor, complement receptor 3 (CR3). For this, we used antibody-coated PANSORBINS® which bound to the ß-chain of CR3 without inducing phagocytosis. We found that these particles elicited an intracellular production of ROM which was dependent on the cytoskeleton and on phospholipase D. In the second study, we showed that the putative calcium-sensor synaptotagmin II is present in neutrophils and is involved in phagocytosis. Synaptotagmin II was found on the specific granules and translocated to the phagosome in a calcium-dependent manner during eR-mediated phagocytosis and to the plasma membrane after stimulation with the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine. In the third study, we demonstrate the presence of synaptotagmin IV in human macrophages. Synaptotagmin IV translocated transiendy to macrophage phagosomes during eR- and FcγR-mediated phagocytosis. We also found that eR- and FcyR-mediated uptake was calcium dependent in these cells. In the fourth study, we show that lipophosphoglycan (LPG) from Leishmania donovani induced elevated levels of periphagosomal F-actin, inhibition of phagolysosome maturation and diminished production of ROM in neutrophils during eR-mediated phagocytosis. Together, our data show that generation of ROM occurs early during eR-mediated phagocytosis and could be involved in intracellular signalling, that synaptotagmins are present in professional phagocytes and could act as calcium sensors in phagosomal maturation and secretion, and that LPG can be used as a tool to investigate how actin can regulate phagosomal maturation in neutrophils.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 64 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 818
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26661Local ID: 11227ISBN: 91-7373-509-4 (print)OAI: oai:DiVA.org:liu-26661DiVA: diva2:247210
Public defence
2003-11-06, Aulan, Hälsans Hus, Universitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-17Bibliographically approved
List of papers
1. Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
Open this publication in new window or tab >>Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
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1999 (English)In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1452, no 2, 133-144 p.Article in journal (Refereed) Published
Abstract [en]

Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the β2-integrins, i.e. anti-CD11b (the α subunit of CR3) and anti-CD18 (the β subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the α subunit of LFA-1) and to a lesser extent anti-CD11c- (the α subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of β2-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26025 (URN)10.1016/S0167-4889(99)00123-8 (DOI)10478 (Local ID)10478 (Archive number)10478 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Synaptotagmin II could confer Ca2+ sensitivity to phagocytosis in human neutrophils
Open this publication in new window or tab >>Synaptotagmin II could confer Ca2+ sensitivity to phagocytosis in human neutrophils
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2002 (English)In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1590, no 1-3, 159-166 p.Article in journal (Refereed) Published
Abstract [en]

Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26522 (URN)10.1016/S0167-4889(02)00209-4 (DOI)11081 (Local ID)11081 (Archive number)11081 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
3. Complement- and IgG-mediated phagocytosis in human macrophages in calcium dependent and involves synoptotagmin IV
Open this publication in new window or tab >>Complement- and IgG-mediated phagocytosis in human macrophages in calcium dependent and involves synoptotagmin IV
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Calcium regulates membrane fusion events during phagolysosome formation in neutrophil granulocytes and vesicle fusion at the neural synapse. Calcium is also required for uptake of IgG-opsonised particles by human neutrophils. The role of calcium during macrophage phagocytosis is less clear. Here we show that phagocytosis of IgG- or serum-opsonised prey is strictly calcium dependent in human monocyte-derived macrophages. We also show the presence and involvement of synaptotagmin II and IV in human macrophages and in the murine macrophage cell line J774. Synaptotagmin IV displayed a granular distribution in resting human macrophages with some translocation to the plasma membrane. Synaptotagrnin IV did not eo localise with the nucleus, the endoplasmic reticulum or the Golgi apparatus. During phagocytosis of IgG- or serum-opsonised prey we observed a distinct, transient translocation of synaptotagmin IV to the phagosome. The kinnetics of synaptotagmin IV translocation was similar to Rab5. LAMP-1, a marker of late endosomes and mature phagolysosomes fused with the phagosome at a later time point. Our results show that complement- and IgG-mediated phagocytosis are dependent on calcium in human macrophages and indicate a role for synaptotagmin IV in the calcium dependent fusion of the phagosome with components of the early endocytic pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84708 (URN)
Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17Bibliographically approved
4. Lipophosphoglycan (LPG) from Leishmania donovani inhibits phagosomal maturation and oxygen redical production in human neutrophils
Open this publication in new window or tab >>Lipophosphoglycan (LPG) from Leishmania donovani inhibits phagosomal maturation and oxygen redical production in human neutrophils
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lipophosphoglycan (LPG) is the major surface glycoconjugate on Leishmania donovani promastigotes. LPG inhibits phagosome maturation and is crucial for parasite survival in macrophages. Fusion of vesicles with the phagosome is essential for the formation of a mature phagolysosorne and depolymerization of periphagosomal F-actin is likely a prerequisite for vesicle fusion. In macrophages LPG induces an accumulation of periphagosomal F-actin which is correlated to inhibition of vesicle fusion to the phagosome. In this work we investigated the effects of LPG on phagosome maturation in human neutrophils. We found that ingestion of serum-opsonised, LPG-coated yeast particles induced increased levels of periphagosomal Factin in neutrophils. Phagosome maturation was studied using antibodies to CD63 (azurophil granules), synaptotagmin II (specific granules) and LAMP-1 (specific granules, secretory vesicles, multivesicular bodies/multilaminar compartments). Results showed impaired translocation of all these three markers to phagosomes containing LPG-coated prey. The translocation of the early endosome marker Rab5A to the phagosome was not affected by LPG. The late endosomal marker Rab7 was not found in human neutrophils. Chemiluminescence studies revealed that serum-opsonised, LPG-coated yeast induced less production of reactive oxygen metabolites (ROM) compared to controls and that the production was mainly intracellular.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84710 (URN)
Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17

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