| 1. |
- Källquist, Linda, et al.
(författare)
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The tetraspanin CD63 is involved in granule targeting of neutrophil elastase.
- 2008
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 112, s. 3444-3454
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Tidskriftsartikel (refereegranskat)abstract
- Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)-positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63 with an adaptor protein-3-dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady state level was similar to wild type in CD63-depleted clones making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63 -depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention and granule targeting of proNE before storage as mature NE.
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| 2. |
- Bauer, Susanne, et al.
(författare)
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Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.
- 2007
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Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 1938-3673 .- 0741-5400. ; 81, s. 458-464
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Tidskriftsartikel (refereegranskat)abstract
- Proteinase 3 (PR3) is found in granules of all nentrophils but also on the plasma membrane of a subset of nentrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of nentrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where A mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera.
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| 3. |
- Ciornei, Cristina, et al.
(författare)
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Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study
- 2006
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Ingår i: BMC Cardiovascular Disorders. - : Springer Science and Business Media LLC. - 1471-2261. ; 6:49
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Death of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis. METHODS: We investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis. RESULTS: The neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 mug/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37. CONCLUSION: This study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis.
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| 4. |
- Gao, Ying, et al.
(författare)
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Granule targeting of soluble tumor necrosis factor (TNF) receptor expressed during granulopoietic maturation in murine bone marrow cells.
- 2006
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Ingår i: European Cytokine Network. - 1952-4005. ; 17:2, s. 98-108
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Tidskriftsartikel (refereegranskat)abstract
- In this experiment, we explored the potential of secretory lysosomes of hematopoietic cells to act as vehicles for immunomodulatory protein delivery at an inflammation site. We investigated whether exogenous soluble TNF-receptor 1 (sTNFR1) could be expressed in primary hematopoietic progenitor cells and become targeted for storage and secretion during granulopoietic differentiation. An sTNFR1 construct with a transmembrane domain (tm) and a cytosol sorting signal (Y) taken from CD63, was retrovirally transduced to lineage-negative murine hematopoietic bone marrow stem/progenitor cells. This process was followed by cytokine-driven granulopoietic maturation. The sTNFR1-tm-Y was found to be synthesized in precursor cells and to persist in mature granulocytes and monocytes/macrophages. Immunofluorescence-localization studies showed a granule pattern of sTNFR1-tm-Y in both precursor and mature granulocytes and secretion to phagosomes after ingestion of bacteria. Immunoelectron microscopy revealed co-localization between the sTNFR1-tm-Y and the primary (azurophil) granule marker myeloperoxidase. Collectively, our results demonstrated granule targeting, storage, and secretion of exogenous sTNFR1-tm-Y constitutively expressed during normal granulopoietic differentiation. These findings support the concept of using storage organelles of circulating hematopoietic cells as vehicles for targeting sites of inflammation with immunoregulatory agents.
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| 5. |
- Gram, Magnus, et al.
(författare)
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The lipocalin alpha1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species.
- 2008
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Ingår i: Free Radical Research. - : Informa UK Limited. - 1029-2470 .- 1071-5762. ; 42:8, s. 725-736
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Tidskriftsartikel (refereegranskat)abstract
- Alpha(1)-microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of alpha(1)-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that alpha(1)-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of alpha(1)-microglobulin was up-regulated by these oxidants and silencing of the alpha(1)-microglobulin expression increased the cytosol oxidation. alpha(1)-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to alpha(1)-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, alpha(1)-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that alpha(1)-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.
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| 6. |
- Lönnbro, Per, et al.
(författare)
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Analysis of neutrophil membrane traffic during phagocytosis
- 2007
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Ingår i: Neutrophil Methods and Protocols (Methods in Molecular Biology). - 1588297888 - 9781588297884
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Bokkapitel (populärvet., debatt m.m.)abstract
- In this chapter, we describe methods to study membrane traffic during phagosome formation and maturation.
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| 7. |
- Lönnbro, Per, et al.
(författare)
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Isolation of bacteria-containing phagosomes by magnetic selection.
- 2008
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Ingår i: BMC Cell Biology. - : Springer Science and Business Media LLC. - 1471-2121. ; 9:June 27
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens. RESULTS: We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes. CONCLUSION: We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation.
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| 8. |
- Nordenfelt, Pontus, et al.
(författare)
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Different Requirements for Early and Late Phases of Azurophilic Granule-Phagosome Fusion
- 2009
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Ingår i: TRAFFIC. - : Wiley. - 1398-9219 .- 1600-0854. ; 10:12, s. 1881-1893
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Tidskriftsartikel (refereegranskat)abstract
- Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on azurophilic granules, we decided to investigate raft recruitment and the signaling requirements for azurophilic granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of azurophilic granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of azurophilic granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.
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| 9. |
- Nordenfelt, Pontus, et al.
(författare)
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Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:10
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion.
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| 10. |
- Olsson, Mattias, 1975-
(författare)
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Role of the CD47/SIRPα-interaction in regulation of macrophage phagocytosis
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- CD47 is a cell surface glycoprotein that is expressed by virtually all cells in the body. Binding of CD47 to the macrophage receptor Signal Regulatory Protein alpha (SIRPα) yields an inhibitory signal that counteracts phagocytosis. Red blood cells (RBCs) that lack CD47 are rapidly cleared from the circulation, whereas CD47 expressing cells have a normal turnover rate. CD47 has therefore been proposed to function as a marker of self, enabling the immune system to discriminate between self and foreign. Thus, the studies of the present thesis aimed at further investigating the role of CD47 as a marker of self in regulating phagocytosis of platelets, phagocytosis of viable or senescent RBCs, and the mechanisms involved. CD47 on platelets was found to regulate their turnover in vivo, since platelets from CD47-/- mice transfused into wild type recipients were cleared more rapidly from the circulation than wild type platelets. In addition, CD47-/- mice were found to suffer from a mild spontaneous thrombocytopenia, without any signs of accelerated platelet apoptosis or increased platelet activation. CD47-/- mice were more sensitive to experimental immune thrombocytopenia (ITP), as compared with wild type mice. In vitro phagocytosis experiments proved that platelet CD47 was responsible for this effect, since blocking antibodies to macrophage SIRPα increased phagocytosis of wild type platelets to the levels seen for CD47-/- platelets. When unopsonized platelets or RBCs from CD47+/- mice (expressing about 50 % less CD47 than wild type cells) were transfused into wild type recipients, they were cleared from the circulation at virtually the same rate as wild type cells. However, CD47+/- cells were cleared more rapidly than wild type cells when transfused animals were challenged with an antibody directed against the transfused cell type. In vitro, IgG-opsonized CD47+/- platelets and RBCs were ingested to a higher extent than wild type cells, but less than CD47-/- cells, suggesting that CD47 dose-dependently regulates phagocytosis in macrophages. It was also investigated if inhibitory SIRPα signaling is localized to the site of contact with the cell that is to be ingested, or whether the inhibition of phagocytosis is more general in the whole macrophage. Experiments with a mix of IgG-opsonized wild type and CD47-/- RBCs showed that the effect of inhibitory CD47-SIRPα signaling was local in the macrophage and limited to the site of contact with a specific target cell. Thus, contact with one or several wild type RBCs did not affect the increased phagocytosis of CD47-/- RBCs by the same macrophage. RBC senescence involves oxidation of membrane lipids and proteins, as well as exposure of phosphatidylserine (PS) on the cell surface, and clearance of senescent RBCs is believed to be regulated by several different factors. To investigate the role of CD47 in uptake of experimentally senescent RBCs, RBCs were oxidized with CuSO4/ascorbic acid (Ox-RBCs). Phagocytosis of Ox-RBCs required recognition of PS on the RBCs, recognition by scavenger receptors on the macrophages, and was strongly dependent on serum. CD47 did not inhibit serum-dependent phagocytosis of experimentally senescent unopsonized RBCs, since phagocytosis of senescent wild type or CD47-/- RBCs was virtually similar. The ability of CD47 to cluster in the plasma membrane upon cross-linking with antibodies was reduced in senescent RBCs. Despite this, CD47 inhibited phagocytosis of IgG-opsonized viable or senescent RBCs to the same extent. In summary, CD47 can function as a marker of self on both RBCs and platelets. The phagocytosis-inhibitory effect is dependent on the CD47 expression level, and CD47-SIRPα signaling acts locally in the macrophage at the contact with a target cell. In experimentally senescent RBCs, CD47 does not inhibit serum-dependent phagocytosis in the absence of opsonization, but still inhibits FcγR-mediated phagocytosis. Key words: CD47, SIRPα, platelets, red blood cells, macrophages, phagocytosis, Fcγ receptor, senescence
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| 11. |
- Pellmé, Sara, 1975, et al.
(författare)
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Localization of human neutrophil interleukin-8 (CXCL-8) to organelle(s) distinct from the classical granules and secretory vesicles
- 2006
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Ingår i: J Leukoc Biol. - : Oxford University Press (OUP). ; 79, s. 564-573
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Tidskriftsartikel (refereegranskat)abstract
- Mature human neutrophils contain small amounts of interleukin-8 [CXC chemokine ligand 8 (CXCL-8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL-8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL-8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL-8 production by lipopolysaccharide. After 8 h of stimulation, CXCL-8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL-8-containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL-8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL-8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL-8 may be localized to the ER or ER-like structures in the neutrophil.
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| 12. |
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| 13. |
- Svartberg, Kenth, et al.
(författare)
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Consistency of personality traits in dogs
- 2005
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Ingår i: Animal Behaviour. - : Elsevier BV. - 0003-3472 .- 1095-8282. ; 69:2, s. 283-291
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the consistency of behaviour over repeated tests in dogs, Canis familiaris. Dogs were tested three times, with an average of 30 and 35 days between tests. The behavioural test used in the study included 10 subtests that exposed dogs to various situations, such as the appearance of an unfamiliar person, play, preylike objects, metallic noise and a suddenly appearing dummy. Studies using the same test with many dogs have revealed five specific personality traits, labelled Playfulness, Chase-proneness, Curiosity/Fearlessness, Sociability and Aggressiveness, and one higher-order, broader dimension, interpreted as a shyness–boldness continuum. We used these traits in the present study. We found significant correlations over the test series in all the specific traits as well as in the Boldness dimension. The magnitude of trait scores for Playfulness, Chase-proneness and Sociability, as well as for the Boldness dimension, was stable between tests. The scores for Aggressiveness and Curiosity/Fearlessness, however, differed between the first two tests: the intensity of behaviour related to fear and aggression decreased from test 1 to test 2, but the intensity of exploratory behaviour increased. This result indicates that these two traits in dogs are sensitive to novelty, although individual differences are also maintained in nonnovel situations. The results suggest that playful, social, exploratory, avoidant and aggressive behaviour in dogs is influenced by stable dispositions; i.e. personality traits, that seem to have been important during the evolution of the domestic dog
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| 14. |
- Tapper, Hans, et al.
(författare)
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Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide.
- 2006
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 312:18, s. 3471-3484
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Tidskriftsartikel (refereegranskat)abstract
- The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta 248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. in addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/ Delta 248-267 expressed in RBL cells bypassed plasma membrane trafficking. our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide. (c) 2006 Elsevier Inc. All rights reserved.
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| 15. |
- Winberg Tinnerfelt, Martin, 1976-
(författare)
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Leukocyte responses to pathogens : integrins, membrane rafts and nitric oxide
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During microbial invasion, leukocytes of the innate immunity are rapidly recruited to the site of infection where they internalize (phagocytose), kill and digest the invaders. To aid this process, leukocytes express surface receptors such as Toll-like receptors, β2-integrins and Fc-receptors. The β2-integrins are also used for attachment to the extracellular matrix and are important for migration. When pro- vs. anti-inflammatory regulation of β2-integrins was investigated, it was found that chemotactic factors modulate neutrophil adhesion through altered affinity and/or avidity of β2-integrins. A bacteria-derived chemoattractant evoked a large increase in affinity as well as in mobility and clustering, while an early, host-derived chemotactic factor induced increased clustering and surface mobility, but only a slight increase in affinity. Anti-inflammatory lipoxin affected β2-integrin avidity, but not affinity.The leukocyte membrane is composed of lipids and proteins, which are inhomogeneously distributed. Specific domains in the membrane, membrane rafts, are enriched in signaling proteins and receptors. It was found that lipophosphoglycan (LPG) a virulence factor and membrane component of the parasite Leishmania donovani, accumulated in macrophage rafts during infection, inhibited PKCα translocation to the membrane and halted phagosomal maturation. Membrane rafts were instrumental for LPG to exert its effect. We further showed that nitric oxide (NO) rescued phagosomal maturation halted by Leishmania donovani parasites, possibly through effects on actin dynamics. NO did not affect parasite virulence per se. Moreover, lipoarabinomannan (LAM), a virulence factor on Mycobacterium tuberculosis (Mtb) bacteria, also inserted itself into macrophage membrane rafts. LAM from a less virulent strain (PILAM) was less efficiently inserted. Insertion could to some extent be inhibited by phosphatidylinositol mannoside (PIM), another structural molecule from Mtb. LAM did not activate the p38 MAPK signaling pathway nor did LAM interfere with TLR 2 or 4 signaling. In neutrophil leukocytes we observed a simultaneous, calciumdependent up-regulation of membrane rafts and secretion of azurophilic granules at the site of phagocytosis. Rafts were also found in the phagosome membrane. Wild type Streptococcus pyogenes bacteria, which can survive phagocytosis, modulated raft delivery.
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