| 1. |
- 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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| 2. |
- Gao, Ying, et al.
(författare)
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Sorting soluble tumor necrosis factor (TNF) receptor for storage and regulated secretion in hematopoietic cells.
- 2004
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Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 1938-3673 .- 0741-5400. ; 76:4, s. 876-885
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoietic cells contain secretory lysosomes that degranulate at sites of inflammation. We envisage that secretory granules can act as vehicles for targeting inflammatory sites, including malignancies, and thereafter, locally release therapeutically active agents to these sites. Exogenous proteins, such as the soluble tumor necrosis factor receptor 1 (sTNFR1), have been shown previously to be targeted to secretory lysosomes [1]. In this work, we asked whether exogenons, secretory lysosome-targeted proteins were subject to regulated secretion. sTNFR1-transmembrane (tm)cytosol-sorting signal (Y) and sTNFR1-tm-Y-enhanced green fluorescent protein (egfp) were expressed in rat basophilic leukemia cell clones having different secretory capacities. sTNFR1-tm-Y was targeted directly from the Golgi to secretory lysosomes, followed by generation of membrane-free sTNFR1, whose secretion could be triggered by a Ca2+ ionophore or inummoglobulin E receptor activation. In contrast, sTNFRI-tm-Y-egfp was targeted to the plasma membrane and then subjected to endocytosis and presumably, secretory lysosome targeting, as judged by results from antibody ligation and cell-surface biotinylation. Activation of protein kinase C with phorbol ester promoted ectodomain shedding at the cell surface, resulting in sTNFR1 release from sTNFR1-tm-Y-egfp. These results support a concept for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.
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| 3. |
- Hansson, Markus, et al.
(författare)
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Biosynthesis, processing, and sorting of human myeloperoxidase.
- 2006
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 445:2, s. 214-224
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Forskningsöversikt (refereegranskat)abstract
- Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states Such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is Unknown, the pro-segirient is required for normal processing and targeting. as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally Occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting. (C) 2005 Elsevier Inc. All rights reserved.
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| 4. |
- Hansson, Markus, et al.
(författare)
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Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.
- 2004
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Ingår i: European Cytokine Network. - 1952-4005. ; 15:3, s. 167-176
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Forskningsöversikt (refereegranskat)abstract
- Cytokines play an important role in the regulation of homeostasis and inflammation. A de-regulated cytokine function can subsequently promote chronic inflammation. This is supported by clinical evidence showing the beneficial effect of inhibiting TNF-alpha through injection of antibodies and soluble receptor in disorders such as rheumatoid arthritis and Crohn's disease. Systemic anti-TNF-alpha therapy however is associated with infectious complications. We therefore suggest a concept for the local deposition of therapeutically active agents into areas of inflammation or malignancy, based on the use of hematopoietic storage and secretory granules as delivery vehicles. Hematopoietic cells are induced to express the therapeutically active protein and to store it in the secretory lysosomes. The cells migrate into a tumour or site of inflammation, where the cells become activated and release the contents of their secretory lysosomes resulting in the local delivery of the therapeutically active protein. In support of this concept, gene transfer and granule loading can be achieved using the soluble TNF-alpha receptor (sTNFR1) after cDNA expression in hematopoietic cell lines. Endoplasmic reticulum (ER)-export can be facilitated by the addition of a transmembrane domain, and constitutive secretion can be prevented by incorporating a cytosol-sorting signal resulting in secretory lysosome targeting. The sTNFR1 is released from the transmembrane domain by proteolytic cleavage and finally, regulated sTNFR1-secretion can be triggered by a calcium signal. In vivo investigations are currently determining the feasibility of local protein delivery at sites of inflammation.
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| 5. |
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| 6. |
- Hansson, Markus, et al.
(författare)
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Targeting proteins to secretory lysosomes of natural killer cells as a principle for immunoregulation.
- 2003
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Ingår i: Molecular Immunology. - 1872-9142. ; 40:6, s. 363-372
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Tidskriftsartikel (refereegranskat)abstract
- Secretory lysosomes of natural killer (NK) cells combine storage, regulated secretion and lysosomal activity. We asked whether one could target exogenous proteins to the secretory lysosomes of NK-cells for final delivery into a tumor site upon degranulation. cDNAs for both soluble and transmembrane (tm) proteins were expressed in the human YT-Indy NK-cell line. Targeting of a soluble TNF receptor (sTNFR1) was achieved by expressing a cDNA construct with a transmembrane sequence to facilitate ER-export and by incorporating a cytosolic sorting signal (Y) from CD63 to overcome constitutive secretion. The resulting sTNFR1-tm-Y was targeted to secretory lysosomes as confirmed by results from biosynthetic radiolabeling in combination with subcellular fractionation, immunoelectron microscopy, and immunofluorescence microscopy. A soluble sTNFR1 form was generated in the secretory lysosome by endogenous proteolytic activity. Expression of exogenous normally secretory non-membrane proteins, such as alpha1-microglobulin (alpha1-m) and alpha1-antitrypsin (alpha1-at) resulted mostly in constitutive secretion although a small amount of alpha1-microglobulin was targeted to secretory lysosomes. Our results suggest a potential for delivery of pharmacologically active agents into tumor sites by use of the NK-cell secretory lysosome as a carrier. (C) 2003 Elsevier Ltd. All rights reserved.
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| 7. |
- Johansson, Åsa, et al.
(författare)
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Secretory lysosome targeting and induced secretion of human soluble TNF-alpha receptor in murine hematopoietic cells in vivo as a principle for immunoregulation in inflammation and malignancy
- 2009
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Ingår i: Experimental Hematology. - : Elsevier BV. - 0301-472X .- 1873-2399. ; 37:8, s. 969-978
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Systemic administration of immunotherapeutics often gives rise to severe side effects. A local deposition, using secretory lysosomes of hematopoietic cells as vehicles for delivery, can overcome this problem. In the present study, the validity of this concept was investigated using retroviral transduction of the human soluble tumor necrosis factor-alpha receptor 1 (hsTNFR1) into murine bone marrow cells, followed by transfer of the genetically modified cells into irradiated mice. MATERIALS AND METHODS: Bone marrow cells from donor mice were transduced with retroviral vector containing cDNA for hsTNFR1, together with a transmembrane domain and a tyrosine-sorting signal in order to facilitate the endoplasmic reticulum export and to achieve secretory lysosome loading. Expression of hsTNFR1 in recipient mice was investigated using flow cytometry and Western blot. Enzyme-linked immunosorbent assay was used to measure levels of tumor necrosis factor-alpha, hsTNFR1, and murine TNFR1. RESULTS: Stable long-term expression of hsTNFR1 was achieved in transplanted mice. Hematopoietic cells, such as natural killer, T and B cells, and neutrophils contained hsTNFR1. Exposure of lipopolysaccaride (in vivo) or phorbole-myristrate esterase (in vitro) induced significant secretion of hsTNFR1. Release of endogeneous murine sTNFR1 did not differ between cells transduced with hsTNFR1 or an "empty" vector. CONCLUSION: Long-term expression in vivo and inducible secretion of hsTNFR1 in murine hematopoietic cells support the potential use of storage organelles in hematopoietic cells as vehicles for targeting inflamed/malignant sites with therapeutically active agents.
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| 8. |
- Källquist, Linda, et al.
(författare)
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Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells.
- 2010
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; Okt, s. 3182-3196
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating "pro(C)"-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.
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| 9. |
- 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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| 10. |
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