Biomedical subjects

C Dahlgren

Publications and source records attributed to C Dahlgren.

At least 37 records · Page 2Linked to original sources

PubMed1999

Respiratory burst in human neutrophils.

During phagocytosis of microbial intruders, professional phagocytes of our innate immune system increase their oxygen consumption through the activity of an NADPH-oxidase that generates superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)). These oxygen metabolites give rise to yet other reactive oxygen species that are strongly anti-microbial but which may also cause damage by destructing surrounding tissue and inducing apoptosis in other immune reactive cells. The development of methodology to measure the generation/release of phagocyte respiratory burst products is thus of great importance, and a number of different techniques are currently in use for this purpose. Three of the techniques that we have used, (luminol/isoluminol amplified chemiluminescence, cytochrome C reduction, and PHPA oxidation technique) are described in more detail in this review. We hope to convince the readers that these techniques are valuable tools in basic as well as more clinically oriented research dealing with phagocyte function. The basic principles for luminol/isoluminol-amplified chemiluminescence is used as the starting point for discussing methodological problems related to measurements of oxygen metabolites generated by professional phagocytes.

Fluorometry↗

PubMed1999

Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.

Antigens, CD↗

PubMed1999

Particles binding beta(2)-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis.

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 beta(2)-integrins, i.e. anti-CD11b (the alpha subunit of CR3) and anti-CD18 (the beta 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 alpha subunit of LFA-1) and to a lesser extent anti-CD11c- (the alpha 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 beta(2)-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.

Antibodies↗

PubMed1999

Cleavage of annexin I in human neutrophils is mediated by a membrane-localized metalloprotease.

A truncated form of annexin I, formed during Ca2+-induced translocation to neutrophil specific granules and secretory vesicles/plasma membranes, is generated through the action of an endogenous membrane protease. The cleavage of annexin I is inhibited by the metalloprotease inhibitor 1,10-phenanthroline as well as by Triton X-100 and dithiothreitol, classifying the protease as a membrane-bound, thiol-dependent metalloprotease. The cleavage site is located close to the N-terminal of annexin I, leaving a truncated form of the molecule, des1-8 annexin I, that contains the Ca2+-binding sites, as well as a number of phosphorylation sites of importance for the function of the protein. When assessing binding capacity to different neutrophil organelles, full-length annexin I bound to azurophil granules, specific granules, and secretory vesicles/plasma membranes, while des1-8 annexin I only bound to specific granules and secretory vesicles/plasma membranes, but not to azurophil granules (C. Sjölin, C. Dahlgren, Biochim. Biophys. Acta 1281 (1996) 227-234). This implies that there are different mechanisms of binding to neutrophil organelles of full-length annexin I and the truncated form, and that cleavage of annexin I might be of regulatory importance for the degranulation process.

Amino Acid Sequence↗

PubMed1999

The synthetic non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline inhibits neutrophil production of reactive oxygen species.

The effects of the non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline (B220) on the generation of reactive oxygen species froin human neutrophils were investigated. The data show that B220 inhibits neutrophil release of reactive oxygen species, as well as intracellular generation of reactive oxygen species. The inhibition is not achieved through direct oxygen radical scavenger activity of B220, and the drug has no immediate effects on the activity of the assembled oxidase. Radical production and release were inhibited by all agonists tested [i.e. the protein kinase C-activating phorbol ester phobol myristate acetate; the receptor-specific agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP); and serum-opsonized yeast particles] in the presence of B220. The drug also inhibits phagocytosis and fMLP-induced mobilization of granules. However, based on the fact that the effects of B220 on phagocytosis and granule mobilization are much less significant than its effect on radical production, we suggest that the signal(s) affected by B220 is (are) located mainly downstream of the point at which the signals are generated to promote oxidase activation and phagocytosis or granule secretion, respectively.

Adult↗

PubMed1999

Stereotactic irradiation of skull base meningiomas with high energy protons.

Nineteen patients with inextirpable skull base meningioma with involvement of neurovascular structures were given irradiation with a 180 MeV proton beam at the The Svedberg Laboratory, Uppsala, Sweden. The patients were treated seated in a fixed position with a stereotactic approach. Titanium-markers to the outer table served for identification and verification of the target positioning for dose planning and irradiation. The patients were given a total dose of 24 Gy in four consecutive daily 6 Gy fractions. All patients have been followed for at least 36 months. So far no meningiomas have progressed after treatment. Two patients have developed corticosteroid responsive oedema in the target area 6 moths after treatment. Late, but not serious, symptoms of side effects have been observed in one patient.

Adult↗

PubMed1999

Activation of human neutrophils by mycobacterial phenolic glycolipids.

The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.

Antigens, Bacterial↗

PubMed1998

Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils.

Galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, was investigated with respect to its ability to activate the human neutrophil NADPH-oxidase. The galectin-3-induced activity was determined with in vivo exudated cells (obtained from a skin chamber) and compared with that of peripheral blood neutrophils. Galectin-3 was found to be a potent activator of the NADPH-oxidase only in exudated neutrophils and the binding of galectin-3 to the surface of these cells was increased compared with peripheral blood cells. Different in vitro priming protocols resulting in degranulation were used to mimic the exudation process in terms of increasing the receptor exposure on the cell surface. Galectin-3 could induce an oxidative response similar to that in exudated cells only after a significant amount of the intracellular organelles had been mobilized. This increase in oxidative response was paralleled by an increased binding of galectin-3 to the surface of the cells. The major conclusion of the study is that galectin-3 is a potent stimulus of the neutrophil respiratory burst, provided that the cells have first experienced an extravasation process. The results also imply that the neutrophil response to galectin-3 could be mediated through receptors mobilized from intracellular granules, and we report the presence of galectin-3-binding proteins in such organelles.

Alkaline Phosphatase↗

PubMed1998

Desensitization of formyl peptide receptors is abolished in calcium ionophore-primed neutrophils: an association of the ligand-receptor complex to the cytoskeleton is not required for a rapid termination of the NADPH-oxidase response.

Binding of ligands to N-formyl peptide chemoattractant receptors exposed on human neutrophils generates signals in the cells that induce an activation of the superoxide anion producing NADPH-oxidase. Ligand binding is followed by a rapid association of the ligand-receptor complex with the cytoskeleton, a process leading to desensitization of the cells with respect to NADPH-oxidase activation. We show that neutrophils that have experienced an intracellular calcium rise obtained through interaction with the calcium-specific ionophore ionomycin are "primed" with respect to the FMLP-induced production of superoxide anions. Mobilization of FMLP receptors from intracellular pools is one well-known mechanism behind the primed response. Based on our finding that ionomycin-treated neutrophils could not be desensitized, we suggest that the lack of association between the ligand-receptor complex and the cytoskeleton is an additional priming mechanism. Since in vivo-exudated neutrophils, which also had mobilized intracellular organelles, could be desensitized, we suggest that the abolished desensitization in ionomycin-treated neutrophils is not due to an inability of newly recruited receptors to couple to the cytoskeleton. We show that a rapid termination of FMLP-induced superoxide anion production is obtained in both desensitizable and nondesensitizable neutrophils, suggesting that the desensitization phenomenon is of limited importance in the oxidase termination process.

Adult↗

PubMed1998

Acupuncture.

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Acupuncture Therapy↗

PubMed1997

Translocation of annexin XI to neutrophil subcellular organelles.

In an earlier study, annexin XI was found to be present in the cytosol of neutrophil granulocytes (Blood (1996) 87, 4817). The protein was isolated by calcium-dependent translocation to specific granules and was found to be a 42-kDa truncated form of annexin XI. Using human autoantibodies directed against annexin XI we have now reinvestigated the ability of full size annexin XI to translocate to different neutrophil organelles isolated by subcellular fractionation. The autoantisera used recognised a protein of 55-kDa in neutrophil cytosol and comparison with a whole cell lysate indicated that the larger portion of the cellular content of this protein is localised to the cytosol. Azurophil granules, specific granules and secretory vesicles/plasma membrane were isolated by subcellular fractionation on Percoll gradients, mixed respectively with neutrophil cytosol and the calcium concentration was raised. Immunoblotting showed that annexin XI translocated to specific granules and secretory vesicles/plasma membrane at 100 micromol/l calcium. When raising the concentration of calcium to 1 mmol/l, annexin XI translocated to the azurophil granules as well. Periphagosomal translocation of annexin XI occurred during phagocytosis of yeast particles, implying that this protein plays a role in the events associated with the phagocytic process.

Annexins↗

PubMed1997

Desensitization of the fMLP-induced NADPH-oxidase response in human neutrophils is lacking in okadaic acid-treated cells.

The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) interacts with neutrophils, generating signals that induce activation of the superoxide anion/hydrogen peroxide-producing NADPH-oxidase. Low temperature binding of fMLP to its neutrophil surface receptors is associated with a desensitization of the cells with respect to activation of the oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself. Furthermore, no desensitization is obtained in the presence of cytochalasin B, suggesting that the cytoskeleton is involved in the process leading to desensitization. Okadaic acid is a toxin produced by dinoflagellates and exerts its effects by an inhibition of cellular phosphatases. To investigate the role of phosphorylation/dephosphorylation events in the desensitization process we used okadaic acid as a scientific tool. We show that neutrophils treated with okadaic acid are primed with respect to the fMLP-induced production of superoxide anion, and that no desensitization is obtained in toxin-treated cells. Because the recovery of ligand-receptor complexes in a Triton X-100-insoluble fraction is very low in the cells treated with okadaic acid, we suggest that protein dephosphorylation is required to obtain binding to the cytoskeleton of occupied fMLP receptors; binding of the occupied receptors to the cell cytoskeleton being the mechanism behind desensitization.

Adult↗

PubMed1997

A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.

Neutrophil production of reactive oxygen species is dependent on an assembly process that involves a translocation of the cytosolic NADPH-oxidase components (p47phox; p67phox; Rac2) to a b cytochrome containing membrane. Based on the fact that an intracellular Ca2+ rise can activate the oxidase without any extracellular release of reactive oxygen species, we suggest that the oxidase can be assembled in a membrane distinct from the plasma membrane. Disintegrated cells were used to monitor Ca2+ dependent membrane binding of neutrophil cytosolic proteins. Membranes containing the b cytochrome part of the oxidase, i.e., specific granules and plasma membranes/secretory vesicles, were used in the translocation experiments. Several cytosolic proteins were found to translocate to specific granules as well as the plasma membranes/secretory vesicles, one of them being annexin I. Using antibodies in the blotting assay against the cytosolic oxidase components p47phox and p67phox, we could show that no Ca2+ dependent translocation of these cytosolic proteins occur to neither of the b cytochrome containing membranes.

Annexin A1↗

PubMed1997

Surface-related triggering of the neutrophil respiratory burst. Characterization of the response induced by IgG adsorbed to hydrophilic and hydrophobic glass surfaces.

Hydrophilic and hydrophobic glass surfaces precoated with human albumin, fibrinogen, or IgG were investigated with respect to their ability to activate the neutrophil NADPH-oxidase. We found that IgG-coated surfaces induced a substantial and prolonged neutrophil production of reactive oxygen species (ROS). When a hydrophilic surface was used to support protein binding, a somewhat lower neutrophil response (around 35%) was obtained, compared with the response induced by IgG on a hydrophobic surface. The production of ROS was completely eliminated when cytochalasin B was added to the measuring system, suggesting the involvement of the cell cytoskeleton in the activation process. The relation between the intra- and extracellular generation of ROS was further assessed, and we found that most of the ROS produced were released from the cells, in agreement with a model in which the activating surfaces induce a 'frustrated' phagocytic response. Serum totally inhibited 'frustrated' phagocytosis provided that the IgG molecules were sticking to a hydrophilic surface.

Albumins↗

PubMed1997

Different expression and mobilisation of the complement regulatory proteins CD35, CD55 and CD59 in neonatal and adult neutrophils.

We investigated the expression and the N-formyl-methionyl-phenylalanine (fMLP)- and interleukin-8 (IL-8)-induced mobilisation of the complement regulatory proteins CR1 (CD35), decay-accelerating factor (CD55) and CD59 on neutrophils in neonates and adults. The expression of CD35 on resting neonatal neutrophils was significantly higher than in adults. Both fMLP and IL-8 increased CD35 expression more markedly in adults. CD55 on resting neutrophils in neonates was significantly higher than in adults, but did not further increase upon fMLP stimulation as opposed to adults. The increase in CD59 expression was more pronounced in adult neutrophils and was significantly higher than in neonates after fMLP activation. The addition of the protease inhibitor phenylmethanesulphonyl fluoride during fMLP activation improved the upregulation of CD35 significantly more in neonates, but not CD55 or CD59. This effect could be replaced by human normal sera. These data demonstrate that differences exist between neonatal and adult neutrophils with respect to the expression and mobilisation of functional receptors related to protection from autologous complement lysis and may indicate differences in the efficiency to circumvent complement-mediated cell damage at the inflammatory site.

Adult↗

PubMed1996

Diverse effects of different neutrophil organelles on truncation and membrane-binding characteristics of annexin I.

A neutrophil annexin I-related protein, detected after translocation of cytosolic proteins to specific granules and secretory vesicles/plasma membrane (Sjölin et al. (1994) Biochem. J. 300, 325-330), has been characterized with respect to origin and organelle-binding properties. The annexin I-related protein is formed as a result of annexin I cleavage, and this occurs during translocation of annexin I to the specific granules and secretory vesicles/plasma membrane, but not when annexin I is translocated to azurophil granules. The cleavage required calcium and it was facilitated in the presence of specific granules or secretory vesicles/plasma membrane, but not in the presence of azurophil granules. We conclude that the membranes of specific granules and secretory vesicles/plasma membrane contain a protease which is able to cleave annexin I into a truncated 38 kDa fragment, which retains the ability to bind to these organelles. The azurophil granules lack the capacity to cleave annexin I as well as the ability to bind the 38 kDa fragment. These findings may implicate a role for annexin I in the divergent regulation of exocytosis of the different neutrophil granules.

Annexin A1↗

PubMed1996

A simple chemiluminescence assay for the determination of reactive oxygen species produced by human neutrophils.

We show that phagocyte production of reactive oxygen species can be measured using a microtitre plate based chemiluminescence blotting technique. The production of reactive oxygen species is determined by their ability to catalyze the oxidation of luminol or isoluminol, resulting in light emission which is recorded on a photographic film. The method permits the determination of NADPH oxidase activity from as few as 9000 cells. It could be used to detect NADPH oxidase defects in neutrophils (e.g. from patients suffering from chronic granulomatous disease), and to screen pharmaceuticals with scavenging activity for reactive oxygen species.

Humans↗

PubMed1996

Isolation by calcium-dependent translation to neutrophil-specific granules of a 42-kD cytosolic protein, identified as being a fragment of annexin XI.

Secretion of neutrophil granules is dependent on calcium, but at the same time this process is regulated differently for each type of granules. We attempted to find calcium-regulated proteins that selectively translocate from the cytosol to the membranes of the neutrophil granules. An in vitro calcium-dependent translocation assay was designed by mixing cytosol with different neutrophil organelles isolated by subcellular fractionation. Immunoblotting using an anti-cytosol antiserum revealed a cytosolic protein of 42 kD that selectively binds to the specific granules of human neutrophils. It was neither associated with the azurophil granules nor with the secretory vesicles/plasma membrane. The protein was translocated at a calcium concentration of 100 micromol/L and binding was further increased by 1 mmol/L calcium. The 42-kD protein was partially purified from neutrophil cytosol by using its affinity for specific granules and by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partly purified protein allowed the 42-kD band to be excised and subjected to tryptic peptide mapping. Peptides from three peaks were N-terminally sequenced. Searching among known proteins, each one of the amino acid sequences was found to share sequence similarity to annexin XI.

Amino Acid Sequence↗