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The role of calcium and calcium-regulated proteins in neutrophil phagocytosis
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophil phagocytosis is an essential component of the innate immunity against invading pathogens. The two types of phagocytosis that are investigated in detail are IgG- and C3bi-mediated phagocytosis. Although the two types are controlled differently, they share the same driving force - reorganisation of the actin cytoskeleton. Subcellular elevations of intracellular free calcium concentration ([Ca2+]1), are critical for this kind of functional response within the neutrophils.

The aim of this study was to try to understand how calcium and certain calciumregulated proteins control phagocytosis in neutrophils, especially the remodelling of the actin cytoskeleton during pseudopod fonnation and the regulation of phagolysosome fusion.

By immunofluorescence staining (IF) and confocal microscopy, we analysed the distribution of Ca2+ stores using antibodies against Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase (SERCA2) and calreticulin, during phagocytosis. The results showed a distinct accumulation of Ca2+ stores around phagosomes and pseudopods. This accumulation is coherent with a local Ca2+ rise seen in the area of phagocytosis and provides a model for how this localised [Ca2+]i is regulated in neutrophils. To further investigate if inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores are involved, we analysed the subcellular distribution of IP3-receptors (IP3Rs), which are located on the ci+ stores. The IP3Rs translocated in a similar manner as did SERCA2 and calreticulin, indicating that the IP3-sensitive Ca2+ stores are involved.

During phagocytosis, an accumulation of phospholipid- and calcium-binding proteins, annexins, can be seen in the periphagosomal area. Several studies have demonstrated that certain annexins promote Ca2+-dependent contact between phospholipid vesicles and/or isolated neutrophil-specific granule membranes. This suggests that annexins, apart from being involved in vesicle aggregation and fusion, participate together with filamentous actin (F-actin) in phagolysosome formation, by establishing a connection between the phagosomal membrane and granule membranes prior to fusion. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. We have, therefore, investigated the role of gelsolin, which is a protein that severs Factin by binding to the barbed ends, and thereby inhibits further polymerisation. The results show that both annexin I and Ill, and gelsolin translocates to the area of phagocytosis, in a Ca2+ -independent manner, where they eo-localise with F-actin.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2000. , 40 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 619
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28584Local ID: 13738ISBN: 91-7219-576-2 (print)OAI: oai:DiVA.org:liu-28584DiVA: diva2:249395
Public defence
2000-03-17, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-02Bibliographically approved
List of papers
1. Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils
Open this publication in new window or tab >>Redistribution of intracellular Ca2+ stores during phagocytosis in human neutrophils
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1994 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 265, no 5177, 1439-1441 p.Article in journal (Refereed) Published
Abstract [en]

Subcellular gradients of cytosolic free Ca2+ concentration, [Ca2+]i, are thought to be critical for the localization of functional responses within a cell. A potential but previously unexplored mechanism for the generation of gradients of [Ca2+]i is the accumulation of Ca2+ stores at the site of Ca2+ action. The distribution of the Ca2+ store markers Ca(2+)-dependent adenosine triphosphatase and calreticulin was investigated in resting and phagocytosing human neutrophils. Both proteins showed an evenly distributed fine granular pattern in nonphagocytosing cells, but became markedly concentrated in the filamentous actin-rich cytoplasmic area around the ingested particle during phagocytosis. This redistribution began at early stages of phagocytosis and did not depend on an increase in [Ca2+]i. Thus, accumulation of Ca2+ stores in a restricted area of the cell may contribute to the generation of localized increases in [Ca2+]i.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79472 (URN)10.1126/science.8073285 (DOI)
Available from: 2012-08-02 Created: 2012-08-02 Last updated: 2017-12-07Bibliographically approved
2. Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
Open this publication in new window or tab >>Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
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1996 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 316, no 1, 137-142 p.Article in journal (Refereed) Published
Abstract [en]

In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79473 (URN)
Available from: 2012-08-02 Created: 2012-08-02 Last updated: 2017-12-07Bibliographically approved
3. Subcellular distribution of annexins, gelsolin and filamentous actin in adherent human neutrophils during phagocytosis
Open this publication in new window or tab >>Subcellular distribution of annexins, gelsolin and filamentous actin in adherent human neutrophils during phagocytosis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Subcellular elevations of cytosolic free calcium concentration ([ea2+];), are critical for certain functional responses within the neutrophil, such asfilamentous actin (F-actin) reorganization and phagolysosome fusion (PLF). During this event, an accumulation of phospholipid- and calciumbinding proteins, annexins, can be seen in the periphagosomal area. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. Gelsolin is a protein that severs F:actin by binding to the barbed ends, and thereby affect further polymerization. In this study, we used immunofluorescence staining and immunogold technique to analyse the distribution of annexin I, annexin III and gelsolin, in relation to the rearrangement ofF-actin during phagocytosos of complement-opsonized yeast particles by adherent human neutrophils. Iu unchallenged cells, both the aunexins and gelsolin were evenly distributed throughout the cells, whereas F-actin was found mostly in the protruding pseudopodia. Upon phagocytosis an accumulation of both. annexin I and annexin III, and gelsolin could be seen w1thm the vicimty of the phagocytic cups and phagosomes where they colocalized with Factin around the ingested particle.

In calcium-depleted cells, the subcellular distributions of annexins and gelsolin were unaffected. On the other hand, there was a total increase inF-actin polymerization.

Our data may indicate that gelsolin is important for the rearrangement of F-actin and that annexin I and annexin III, which are present in high concentrations in neutrophils, may participate in the following calciumdependent PLF in human neutrophils.

Keyword
Annexins, Ca2+, F-actin, gelsolin, phagocytosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79474 (URN)
Available from: 2012-08-02 Created: 2012-08-02 Last updated: 2012-08-02Bibliographically approved
4. Selective Inhibition of IgG-Mediated Phagocytosis in Gelsolin-Deficient Murine Neutrophils
Open this publication in new window or tab >>Selective Inhibition of IgG-Mediated Phagocytosis in Gelsolin-Deficient Murine Neutrophils
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2000 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 165, no 5, 2451-2457 p.Article in journal (Refereed) Published
Abstract [en]

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn neutrophils was reduced (∼50%) but not to the same extent as ingestion (∼73%). This was not due to reduced surface expression of the Fcγ-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-49420 (URN)10946270 (PubMedID)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved

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