Biomedical subjects

C Dahlgren

Publications and source records attributed to C Dahlgren.

At least 55 records · Page 3Linked to original sources

PubMed1996

Identification of the lysosomal membrane glycoprotein Lamp-1 as a receptor for type-1-fimbriated (mannose-specific) Escherichia coli.

The presence of several glycosylated sites with high-mannose oligosaccharides on the lysosome-associated membrane glycoproteins (Lamps)_ combined with the fact that neutrophil Lamps are present in mobilizable organelles inspired us to investigate their ability to bind type-1 fimbriated (mannose-binding) Escherichia coli and subsequently define a potential function for the Lamps in human neutrophils. Bacterial binding to Lamps purified from chronic myeloic leukemia cells was investigated by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transfer to a blotting membrane and overlay with type-1-fimbriated bacteria. The overlays were developed by growth. The bacteria bound readily to Lamp-1 while there was almost no binding to Lamp-2. Hence, we state that a possible function for neutrophil Lamp-1 is bacterial binding.

Antigens, CD↗

PubMed1996

Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told.

The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA-induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule-deficient HL-60 cells and cord blood neutrophils, we suggest that the scavenger-insensitive part of the NADPH oxidase activity in normal neutrophils resides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.

Adult↗

PubMed1996

Okadaic acid inhibits the signal responsible for activation of the NADPH-oxidase in neutrophils stimulated with serum-opsonized yeast.

The effects of the phosphatase inhibitor okadaic acid (OA) on human neutrophil phagocytic activity were investigated. The chemiluminescence response was found to be greatly reduced in OA-treated neutrophils during phagocytosis of serum opsonized yeast particles in comparison to control cells. However, the OA-treated neutrophils phagocytosed yeast particles to the same extent as control cells and the engulfment of the prey was accompanied by phagolysosomal formation in both OA-treated and nontreated cells. We thus found that the OA effect was selective in the sense that it inhibits the NADPH-oxidase activity in neutrophils phagocytosing yeast particles but not uptake of the prey or phagolysosomal formation. Based on the fact that the NADPH-oxidase activity induced by the chemoattractant formyl-methionyl-leucyl-phenylalanine was not reduced but elevated in OA-treated neutrophils, we conclude that the state of phosphorylation has no direct effect on the oxidase, but is of importance for the NADPH-oxidase activating signal(s) generated during phagocytosis of the yeast particles.

Animals↗

PubMed1996

Secretion of type-1-fimbriae binding proteins from human neutrophil granulocytes.

Granule matrix proteins secreted from human neutrophils after ionomycin stimulation were separated by SDS-PAGE, blotted onto a polyvinylidene diflouride (PVDF) membrane and overlaid with the mannose-binding lectin concanavalin A (Con A) or Escherichia coli bacteria exposing type-I-fimbriae. Four proteins of approximately 30, 40, 70 and 80 kD, respectively, derived from both the azurophil and the specific granules were shown to expose mannose-containing structures by binding of Con A. Such reactivity was also shown for a 90-kD protein from the light membrane fraction enriched in plasma membrane and secretory vesicles. When blots of granule matrix proteins were exposed to type-I-fimbriated bacteria, a total of seven proteins was recognized; four of the five Con A-binding proteins (40, 70, 80 and 90 kD) was detected also by the bacteria in addition to three proteins not detected by Con A (50, 60 and 100 kD). The role of the secreted type-1-fimbriae binding proteins as anti-adhesin candidates is discussed.

Adult↗

PubMed1996

Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils.

A very sensitive chemiluminescence (CL) method was developed for the determination of respiratory burst products generated by the NADPH-oxidase in human neutrophils. Despite the fact that the CL reaction is peroxidase dependent, hydrogen peroxide was found not to participate in the light generating reaction. Phagocytic cells were mixed with isoluminol, a chemiluminescence substrate that detects extracellularly released oxygen species only. Owing to the fact that the availability of released cellular myeloperoxidase is a limiting factor in the reaction, extra peroxidase (horseradish peroxidase; HRP) has to be added to the measuring system. From the fact that the response to phorbol myristate acetate (PMA), as well as to formyl-methionyl-leucyl-phenylalanine (fMLP) was almost totally inhibited by superoxide dismutase (SOD), we conclude that O2.- is the reacting oxygen species measured. The assay system described is well suited for real-time studies of superoxide anion release from activated neutrophils. With the technique, the release of O2.- can be detected from as few as 250 neutrophils.

Catalase↗

PubMed1996

Neutrophils in mucosal secretion are functionally active.

The leucocytes in the secretion on the tonsillar surface have been regarded as inactivated and dying cells with no essential function in the defence of the underlying mucosa. In recent studies, we showed that in acute tonsillitis there are very few, if any, bacteria in the parenchyma, whereas in the secretion on the tonsillar surface there are huge numbers of bacteria and neutrophils, and furthermore, extensive phagocytosis. The present study was performed to elucidate the functional capacity of the neutrophils in the secretion on the tonsillar surface. Secretion samples were taken from the tonsillar surface of healthy volunteers with an imprint technique which allows transfer of secretion from the mucosa to a glass slide with maintained topographic position of the cells. The capacity of the neutrophils to respond to chemotactic stimuli and to phagocytize and further process labelled yeast particles was studied in this in vitro system. The results show that the neutrophils in the secretion have the same properties as in tissue, and also when having arrived in the secretion they can identify and attack new prey.

Adult↗

PubMed1995

The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils.

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

Antibodies, Monoclonal↗

PubMed1995

Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils.

The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.

Alkaline Phosphatase↗

PubMed1995

Detection of glycoprotein receptors on blotting membranes by binding of live bacteria and amplification by growth.

Conditions have been adapted for detecting bacteria bound to glycoprotein receptors on blotting membranes using a self-enhancing detection method based on bacterial growth. Neutrophil plasma membrane proteins, mediating adherence of mannose-binding type-1-fimbriated Escherichia coli and concanavalin A (Con A) to intact human neutrophils, were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was immersed in a suspension of mannose-binding type-1-fimbriated E. coli, and after repeated washings, bound bacteria were allowed to multiply into bacterial colonies by placing the membrane on a solid nutrient substratum. About one major and eight minor glycoproteins, some of which also were detected by Con A, selectively induced colony formation in a mannose-inhibitable fashion. Binding of [35S]methionine metabolically labeled E. coli to PVDF membranes produced a virtually identical binding pattern, demonstrating further the accuracy of this self-enhancing detection method which is rapid, simple, and sensitive and avoids radioisotopes.

Bacterial Adhesion↗

PubMed1995

Different subcellular localization of cytochrome b and the dormant NADPH-oxidase in neutrophils and macrophages: effect on the production of reactive oxygen species during phagocytosis.

When neutrophils and macrophages phagocytose a prey, e.g., complement (C3b)-opsonized yeast particles, the oxygen radical generating NADPH-oxidase is activated. In neutrophils, most of the production of oxygen metabolites occurred in an intracellular compartment, possibly in the phagolysosome. In contrast, no intracellular production could be detected in human macrophages. In these cells, the subcellular localization of the superoxide-generating NADPH-oxidase and associated cytochrome b was assessed in intact cells with indirect immunofluorescence and confocal laser scanning microscopy, and with subcellular fractionation, using centrifugation on Percoll density gradients. A dual localization of the cytochrome b as well as the dormant NADPH-oxidase activity in neutrophils was in agreement with earlier immunocytochemical, biochemical, and subcellular fractionation studies. Furthermore, most of the activity was recovered from the specific granules, whereas only a small fraction was retained in the plasma membrane. In contrast, the cytochrome b/NADPH-oxidase activity in macrophages localized primarily in the plasma membrane fraction. We suggest that the macrophages are incapable of producing reactive oxygen species intraphagosomally, due to an absence of a granule-localized pool of the membrane components of the NADPH-oxidase.

Adult↗

PubMed1995

Chemoattractant-induced NADPH oxidase activity in human monocytes is terminated without any association of receptor-ligand complex to cytoskeleton.

When the chemotactic peptide formylmethionyl-leucyl-phenylalanine binds to its cell surface receptor, a transmembrane signal is generated that activates the superoxide-producing NADPH oxidase of human phagocytes. Comparing monocytes and neutrophils with regard to the production of superoxide anion induced by the peptide, we found a similar time-course for both types of cells. In neutrophils, ligand binding induced a conversion of the receptor to a high-affinity form, a change suggested to be due to an association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton. This event has been hypothesized to terminate the signal that activates the NADPH oxidase and thereby results in cessation of the cellular production of superoxide anion. Neutrophils preincubated with the cytoskeleton-disrupting drug cytochalasin B showed an increased and prolonged superoxide anion production after activation with the peptide, thus indicating that the cytoskeleton is involved in terminating this response. Formylmethionyl-leucyl-phenylalanine was also found to induce polymerization of actin in monocytes; however, cytochalasin B had no effect on the peptide-induced generation of superoxide anion in these cells. Furthermore, also in monocytes, ligand binding induced a conversion of the receptor to a high-affinity form; however, the receptor-ligand complex did not coisolate with the Triton X-100-insoluble cytoskeleton. These results indicate that, in monocytes, the NADPH oxidase activating pathway is terminated without any association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton.

Actins↗

PubMed1995

The serine protease inhibitor diisopropylfluorophosphate inhibits neutrophil NADPH-oxidase activity induced by the calcium ionophore ionomycin and serum opsonised yeast particles.

The effect on human neutrophil NADPH-oxidase activity of the serine protease inhibitor diisopropylfluorophosphate (DFP) was investigated. Pretreatment of neutrophils with the protease inhibitor did not affect the release of reactive oxygen species induced by fMLP. However, the intracellular production of reactive oxygen species induced by ionomycin and yeast particles was largely inhibited in DFP treated cells. Production of reactive oxygen species in subcellular fractions was not affected by the protease inhibitor, neither when the plasma membrane nor when the specific granules were used as source for the b cytochrome subunit of the oxidase. This shows that DFP does not affect the assembly of the oxidase or the activity of the assembled complex. We suggest that serine protease activity is of importance for the signal(s) induced by the Ca2+ ionophore and the yeast particles to reach the dormant NADPH oxidase present in the specific granules and phagolysosomes.

Adult↗

PubMed1995

Serum protects against azurophil granule dependent down-regulation of complement receptor type 1 (CR1) on human neutrophils.

We have investigated the effect of gradual degranulation on the expression of functional receptors (CR1 and CR3) on human neutrophils. Incubation with increasing concentrations of fMLP (10(-10) - 10(-7) M) translocated CR1 and CR3 to the cell surface in a similar kinetic pattern. When reaching maximal expression of receptors (10(-7) M fMLP), 78 +/- 10% and 87 +/- 9% of the total pool of CR1 and CR3, respectively, were translocated to the cell surface. To drive the mobilization process further, cytochalasin B was introduced to increase the stimulatory effect of fMLP. No further increase in CR1 surface expression was obtained. However, we found a characteristic time course of surface appearance of CR1 and CR3 with a maximal surface expression within 1 minute, followed by a time-related down-regulation of CR1 but not CR3. In addition, the total pool of CR1 in cytochalasin B treated neutrophils was reduced after 15 minutes stimulation with fMLP measured by flow cytometry and immunoblotting, indicating degradation of CR1. The down-regulation of CR1 was concomitant with a translocation of azurophil granules, in terms of upregulation of CD63. Azurophil, but not specific nor secretory, granule fractions caused a down-regulation of CR1 on fMLP activated neutrophils. The presence of human sera and serine protease inhibitor protected CR1 from down-regulation. Together, these findings indicate that intracellular stored proteases, released in the late part of the sequential mobilization process, alters the expression of functional receptors mobilized in the early part of the mobilization process. The findings also focus on the importance of the microenvironment for the net outcome of neutrophil activation in terms of functional receptor expression.

Adolescent↗

PubMed1995

Lack of correlation between NADPH-oxidase priming and elevated alkaline phosphatase activity in cord blood neutrophils.

Human neutrophils were isolated from cord blood drawn after Caesarean section deliveries without labour and from peripheral blood from healthy adults. The alkaline phosphatase (ALP) activity in the cell populations was compared with the release of respiratory burst products after activation with the chemoattractant n-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to cord blood neutrophils obtained from babies after normal vaginal deliveries, the Caesarean section neutrophils were not metabolically primed. However, like the neutrophils from vaginally delivered babies, the Caesarean section neutrophils showed an increased ALP activity compared to adult control neutrophils. These findings indicate that priming of cord blood neutrophils appears to be induced by labour and can be dissociated from increased ALP activity.

Adult↗

PubMed1995

Escherichia coli-induced activation of neutrophil NADPH-oxidase: lipopolysaccharide and formylated peptides act synergistically to induce release of reactive oxygen metabolites.

The prevailing view of neutrophil NADPH-oxidase activation during interaction with bacteria is that the production of toxic oxygen metabolites should be directed into the phagosome containing the engulfed prey. However, in this report we show that a common Escherichia coli strain, HB101, may induce a release of neutrophil oxygen metabolites to the extracellular milieu. This phenomenon is dependent on three factors: (i) the mobilization (upregulation) of neutrophil secretory vesicles prior to interaction with the bacteria, (ii) soluble bacterial factors binding to the formylmethionyl-leucyl-phenylalanine receptor and tentatively identified as formylated peptides, and (iii) a bacterium-associated priming factor identified as lipopolysaccharide.

Adult↗

PubMed1995

Neutrophil alkaline phosphatase activity increase in bacterial infections is not associated with a general increase in secretory vesicle membrane components.

The content of alkaline phosphatase (ALP) was determined in neutrophils isolated from patients with acute bacterial infections by a standard enzyme assay. Compared with control cells, patient cells exhibited about a fivefold increase in ALP activity. There was no difference between the ALP Km values of control and patient cells, which indicates that the elevated activity in patient cells was due to the presence of increased amounts of the enzyme. The ALP isozyme in both cell types was determined to be the tissue-unspecific ALP. The fact that much of the ALP activity was measurable only in the presence of detergent suggested that the enzyme was localized in the secretory vesicles, a putative reservoir of plasma membrane components. The amount and subcellular distribution of two other secretory vesicle membrane proteins, i.e., cytochrome b and complement receptor 3, were not altered; hence, we conclude that there was no general increase in amounts of secretory vesicle membrane constituents in the patient cells.

Alkaline Phosphatase↗

PubMed1994

Histaminergic regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites.

Monocytes, recovered by centrifugal elutriation, effectively inhibit functions of human NK cells in vitro. Histamine, acting via monocyte H2-type histamine receptors, abrogates the inhibitory signal. The aim of this study was to define the histamine-reversible mechanism by which monocytes inhibit NK cells with special reference to the respiratory burst activity of monocytes. Monocytes recovered from patients with chronic granulomatous disease did not suppress NK cell function, indicating the requirement of intact nicotinamide-adenine dinucleotide phosphate (NAPDH) oxidase activity of monocytes to inhibit NK cells. Furthermore, catalase, a scavenger of hydrogen peroxide (H2O2), was found to potently reverse the monocyte-induced inhibition of NK cell function; on the other hand, superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, the hypochlorous acid scavenger taurin, or the nitric oxide synthetase inhibitor NG-monomethyl-L-arginine (L-NMMA) did not affect the monocyte-derived, suppressive signal. H2O2, at micromolar concentrations, reconstituted the inhibitory effects of monocytes on NK cell function. Histamine had no scavenger activity but effectively suppressed the generation of H2O2 in isolated monocytes. This effect of histamine was transduced by H2-type histamine receptors. We conclude that the histamine-reversible, inhibitory effect of elutriated monocytes on NK cells is dependent on the formation of reactive oxygen metabolites by monocytes and that H2O2 is a pivotal mediator of the suppressive signal.

Cytotoxicity Tests, Immunologic↗

PubMed1994

Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: effect of an association of the ligand-receptor complex to the cytoskeleton.

The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15 degrees C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-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, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.

Calcium↗