PubMed Health⌕ Search

Biomedical subjects

Hans Tapper

Publications and source records attributed to Hans Tapper.

At least 19 recordsLinked to original sources
PubMed2006

Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study.

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.

Animals↗
PubMed2006

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (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 neutrophils 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 all 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.

Cell Membrane↗
PubMed2006

Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide.

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/Delta248-267, 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/Delta248-267 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.

Animals↗
PubMed2006

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes.

We recently reported that the human pathogen Streptococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal molecules from their azurophilic and specific granules into phagosomes, can be modulated by S. pyogenes bacteria expressing surface-associated M and/or M-like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild-type S. pyogenes, after internalization by human neutrophils, inhibits the fusion of azurophilic granules with phagosomes. In contrast, azurophilic granule-content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M-like proteins. Also, when heat-killed wild-type bacteria are used as the phagocytic prey, fusion of azurophilic granules with phagosomes is observed. The inhibition caused by live wild-type S. pyogenes is specific for azurophilic granule-phagosome fusion, because the mobilization of specific granules and the production of reactive oxygen species are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M-like proteins selectively prevent the fusion of azurophilic granules with phagosomes.

Azure Stains↗
PubMed2006

Granule targeting of soluble tumor necrosis factor (TNF) receptor expressed during granulopoietic maturation in murine bone marrow cells.

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.

Animals↗
PubMed2005

Localization of human neutrophil interleukin-8 (CXCL-8) to organelle(s) distinct from the classical granules and secretory vesicles.

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.

Biomarkers↗
PubMed2004

Membrane retrieval in neutrophils during phagocytosis: inhibition by M protein-expressing S. pyogenes bacteria.

During phagocytosis and phagosome maturation, complex membrane traffic events must be coordinated. We have observed, using fluorescent fluid-phase and membrane markers, that in the human neutrophil, internalization of nonopsonized, Gram-positive bacteria, but not of latex beads, is accompanied by a rapid and localized formation of pinosomal structures. This pinocytic response is calcium-dependent but insensitive to actin cytoskeleton disruption and wortmannin treatment. Contrary to what we observe, endosomal structures usually are considered to participate in phagosome formation by providing necessary membrane to forming phagosomes. Instead, our results show a coupling between neutrophil secretory and membrane-retrieval processes during phagosome maturation, and we suggest that the observed, localized pinocytic response is linked to the secretion of azurophilic granules toward nascent phagosomes. Accordingly, M and M-like protein-expressing Streptococcus pyogenes bacteria, which are able to survive inside neutrophil phagosomes, inhibit both the secretion of azurophilic granules to phagosomes and pinosome formation.

Antigens, Bacterial↗
PubMed2004

Sorting soluble tumor necrosis factor (TNF) receptor for storage and regulated secretion in hematopoietic cells.

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 exogenous, 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 immunoglobulin E receptor activation. In contrast, sTNFR1-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.

Animals↗
PubMed2004

Human eosinophils produce the T cell-attracting chemokines MIG and IP-10 upon stimulation with IFN-gamma.

Eosinophils participate in allergic inflammation, where expression of T helper cell type 2 (Th2) cytokines such as interleukin (IL)-4 and IL-5 are seen. However, eosinophils sometimes accumulate during disease with expression of Th1 cytokines [i.e., interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-1beta]. In this study, we investigated whether eosinophils can respond with expression of the IFN-inducible C-X-C chemokines monokine induced by IFN-gamma [MIG; CXC chemokine ligand 9 (CXCL9)], IFN-gamma-inducible protein (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11). These chemokines share the ability to recruit and activate T cells and natural killer cells to sites of inflammation. We found that IFN-gamma induced rapid and sustained gene expression of MIG, IP-10, and I-TAC in eosinophils, as detected by quantitative reverse transcriptase-polymerase chain reaction. During incubation, IFN-gamma-stimulated eosinophils released MIG and IP-10, as detected by enzyme-linked immunosorbent assay, while I-TAC could not be detected in the medium. TNF-alpha but not IL-1beta enhanced the IFN-gamma-induced production of MIG and IP-10. Conversely, addition of the Th2 cytokine IL-4 down-regulated IFN-gamma-induced synthesis of MIG and IP-10 in eosinophils. Crohn's disease is characterized by a Th1-polarized inflammation and presence of eosinophils. In lesions from this disease, MIG was detected in eosinophils by immunohistochemistry. Taken together, the results point to immunoregulatory roles for eosinophils during some diseases with Th1-polarized inflammation.

Cell Line↗
PubMed2003

Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.

Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.

Amino Acid Sequence↗
PubMed2003

Targeting proteins to secretory lysosomes of natural killer cells as a principle for immunoregulation.

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.

Animals↗
PubMed2003

Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth.

Leg ulcers of venous origin represent a disease affecting 0.1-0.2% of the population. It is known that almost all chronic ulcers are colonized by different bacteria, such as staphylococci, enterococci and Pseudomonas aeruginosa. We here report that P. aeruginosa, expressing the major metalloproteinase elastase, induces degradation of complement C3, various antiproteinases, kininogens, fibroblast proteins, and proteoglycans (PG) in vitro, thus mimicking proteolytic activity previously identified in chronic ulcer fluid in vivo. Elastase-producing P. aeruginosa isolates were shown to significantly degrade human wound fluid as well as human skin proteins ex vivo. Elastase-containing conditioned P. aeruginosa medium and purified elastase inhibited fibroblast cell growth. These effects, in conjunction with the finding that proteinase production was detected in wound fluid ex vivo, suggest that bacterial proteinases play a pathogenic role in chronic ulcers.

Apoptosis↗
PubMed2003

Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils.

Strains of the Gram-positive human pathogen Streptococcus pyogenes (group A streptococcus) that express surface-associated M or M-like proteins survive and grow in non-immune fresh human blood. This is generally accepted to be caused by an antiphagocytic property of these proteins. However, in most previous studies, an inhibition of the internalization of the bacteria into host cells has not been studied or not directly demonstrated. Therefore, in the present paper, we used flow cytometry, fluorescence microscopy and electron microscopy to study phagocytosis by human neutrophils of wild-type S. pyogenes and strains deficient in expression of M protein and/or the M-like protein H. The results demonstrate that all strains of S. pyogenes tested, including the wild-type AP1 strain, induce actin polymerization and are efficiently phagocytosed by human neutrophils. In addition, using classical bactericidal assays, we show that the wild-type AP1 strain can survive inside neutrophils, whereas mutant strains are rapidly killed. We conclude that the ability of virulent S. pyogenes to survive and multiply in whole blood is most likely not possible to explain only by an antiphagocytic effect of bacterial surface components. Instead, our data suggest that bacterial evasion of host defences occurs intracellularly and that survival inside human neutrophils may contribute to the pathogenesis of S. pyogenes and the recurrence of S. pyogenes infections.

Actins↗
PubMed2002

Fc gamma R-mediated phagocytosis stimulates localized pinocytosis in human neutrophils.

Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcgammaR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcgammaR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca(2+) concentration were prevented. Because stimulation of FcgammaR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcgammaR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca(2+) dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcgammaR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation.

Calcium↗
PubMed2002

Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules.

The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phagosomes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome.

Actins↗
PubMed2002

Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles.

Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of azurophilic granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized azurophilic granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability.

Adult↗