Search PubMed⌕ Search

Biomedical subjects

N Borregaard

Publications and source records attributed to N Borregaard.

At least 109 records · Page 6Linked to original sources
PubMed1992

Human neutrophil gelatinase: a marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay.

Human neutrophil gelatinase was purified to apparent homogeneity. The N-terminal amino-acid sequence of the purified enzyme could be aligned to an internal part of the cDNA-derived amino-acid sequence of 92-kDa type IV collagenase from SV 40-transfected human lung fibroblasts and from a TPA differentiated monocytic cell line, U937. Total amino-acid composition of U937 and neutrophil gelatinases was identical. Gelatinase was susceptible to treatment with o- and n-glycanase, indicating that posttranslational addition of oligosaccharide side chains occurs. An enzyme-linked immunosorbent assay for gelatinase was developed using specific polyclonal rabbit antibodies. The assay was specific, sensitive, accurate, and reproducible. Ninety percent range for plasma gelatinase from normal subjects was 17.3 to 102.9 ng/ml. In patients treated with cytostatic agents for non-Hodgkin's lymphoma, there was a parallel drop in plasma gelatinase and peripheral granulocyte count. This indicates that plasma gelatinase is a marker for circulating neutrophils. Plasma gelatinase does not seem to reflect bone marrow cellularity.

Amino Acid Sequence↗
PubMed1992

Stimulus-dependent secretion of plasma proteins from human neutrophils.

In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli.

Blood Proteins↗
PubMed1991

Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma.

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit the chemotaxis and enhance the oxidative burst response of human neutrophils in vitro. The present study describes the effect of recombinant GM-CSF on the neutrophil and monocyte function in patients with lymphoma undergoing GM-CSF treatment. Patients with either Hodgkin's or non-Hodgkin's lymphoma were treated with various dosages (2-16 micrograms kg-1 body weight per day for 5 days) of rhGM-CSF by intravenous or subcutaneous route. Prior to and on day 5 of rhGM-CSF treatment, neutrophil and monocyte chemotaxis and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence system may be enhanced by GM-CSF treatment and that this cytokine may be a useful therapeutic adjunct in compromised patients.

Cell Degranulation↗
PubMed1990

Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine.

Alkaline Phosphatase↗
PubMed1990

Neutrophil beta-2 microglobulin: an inflammatory mediator.

Beta-2 microglobulin (beta 2m) constitutes the light invariant chain of HLA class I antigen, and is a constituent of mobilizable compartments of neutrophils. Two forms of beta 2m exist: native beta 2m and proteolytically modified beta 2m (Des-Lys58-beta 2m), which shows alpha mobility in crossed radioimmuno-electrophoresis. The modification of native beta 2m can be executed by membrane-associated activity of mononuclear cells, and Des-Lys58-beta 2m augments the production of interleukin 2. In this study we present evidence that human neutrophils contain native beta 2m in specific granules, secretory vesicles, and plasma membrane. Beta 2m was released in the native form from neutrophils in response to stimulation with chemotactic stimuli and phorbol ester. The results of experiments designed to study the modification of native beta 2m by neutrophils indicated that neutrophils do not participate in the proteolysis of beta 2m. However, we demonstrated that native beta 2m following degranulation may be transformed to Des-Lys58-beta 2m by lymphocytes. We suggest that neutrophil beta 2m following exocytosis may be transformed to Des-Lys58-beta 2m, acting as an extracellular messenger between granulocytes and lymphocytes in the inflammatory focus.

Adolescent↗
PubMed1990

Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase.

Tetranectin, a protein recently identified in a wide variety of human secretory cells (Christensen, L., and I. Clemmensen. 1989. Histochemistry. 92:29-35) was found to colocalize with latent alkaline phosphatase activity in fractions well separated from azurophil granules, specific granules, gelatinase-containing granules, and plasma membranes when postnuclear supernatants of nitrogen-cavitated neutrophils were fractionated on discontinuous Percoll density gradients. Stimulation of intact neutrophils with nanomolar concentrations of FMLP, leukotriene B4, 10-100 U/ml of tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor resulted in parallel release of tetranectin and translocation of alkaline phosphatase to the plasma membrane. Furthermore, intracellular pools of tetranectin and latent alkaline phosphatase were completely released from neutrophils under conditions that barely induced release of specific granules containing B12-binding protein. These findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules. These organelles may play an important role as stores of membrane proteins that are mobilized to the cell surface during stimulation by inflammatory mediators.

Alkaline Phosphatase↗
PubMed1989

Quantitative analysis of phospholipids and demonstration of plasmalogen in human neutrophil subcellular fractions by high-performance liquid chromatography.

The subcellular distribution of phospholipids in unstimulated neutrophils was investigated by high-performance liquid chromatography of lipid extracts of granule and plasma membranes obtained from Percoll density gradients. The mobile phase contained acetonitrile:methanol:85% phosphoric acid (131:3:0.8 v/v/v). Post-nuclear supernatants contained 2.3 micrograms lipid phosphorus/mg protein. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine were demonstrated in azurophil granules, specific granules and in a combined fraction of secretory granules and plasma membranes. Separate estimates of each phospholipid class by peak areas obtained by high-performance liquid chromatography showed that secretory granules and plasma membranes contained most of the phosphatidylinositol and phosphatidylcholine (p less than 0.005 vs peaks areas obtained in azurophil and specific granules), whereas a major part of the phosphatidylethanolamine was located in the specific granules (p less than 0.005 vs peak areas obtained in azurophil granules, and plasma membrane and secretory granules). High-performance liquid chromatography proved to be a useful principle for the demonstration of plasmalogen because the acidic solvent caused hydrolysis of phosphatidalethanolamine, which was recovered as lysophosphatidylethanolamine. Additionally, sphingomyelin was demonstrated in all subfractions by thin-layer chromatography.

Cell Membrane↗
PubMed1989

Translocation of protein kinase C to subcellular fractions of human neutrophils.

The subcellular localization of protein kinase C in unstimulated human neutrophils and neutrophils stimulated by phorbol-myristate-acetate (PMA), 1-oleoyl-2-acetyl-rac-glycerol (OAG), and ionomycin was investigated in subcellular fractions obtained by nitrogen cavitation and Percoll density gradient centrifugation. Protein kinase C was found to be localized mainly in the cytosol in unstimulated cells, whereas significant translocation to fractions containing the plasma membrane was observed after stimulation by PMA, OAG, and ionomycin. At the same time, phospholipid-insensitive protein kinase activity appeared in the cytosol and the plasma membrane fractions. To determine whether binding of protein kinase C occurred to the plasma membrane or to intracellular membranes that had translocated to the plasma membrane, we investigated the ability of isolated azurophil, specific and secretory granules, and plasma membrane vesicles to bind protein kinase C in response to addition of PMA and OAG. Only fractions containing plasma membranes and secretory granules were able to bind protein kinase C. The observation explains the selective activation of plasma membrane structures by protein kinase C.

Binding Sites↗
PubMed1989

Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils.

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.

Alkaline Phosphatase↗
PubMed1988

Subcellular localization and dynamics of components of the respiratory burst oxidase.

Membrane and cytosolic factors cooperate to generate NADPH-oxidase. The study of the syndrome of NADPH-oxidase deficiencies, chronic granulomatous disease, has enabled the identification of two membrane factors: a flavin adenine dinucleotide flavoprotein and a b cytochrome. The nature of the cytosolic components is still unknown, but a 47-kD protein, whose phosphorylation occurs in parallel with the generation of a respiratory burst in intact cells, seems to be one of the cytosolic factors. The subcellular localization of the membrane-bound NADPH-oxidase components has been studied in neutrophils: In unstimulated cells, only a minute fraction of the NADPH-oxidase components is localized in the plasma membrane, whereas approximately 80% is localized in the membrane of the specific granules and the majority of the rest is in a newly described membrane-bound compartment, the secretory granules, identified by latent alkaline phosphatase. During stimulation, these NADPH-oxidase components are translocated to the plasma membrane as a result of fusion of granule membrane with plasma membrane. Only the NADPH-oxidase components present in the plasma membrane are incorporated in the respiratory burst oxidase generated in intact cells.

Animals↗
PubMed1988

Lactoferrin-mediated transfer of iron to intracellular ferritin in human monocytes.

Interactions of 125I-59Fe-lactoferrin with human monocytes were studied. After 4 hours of incubation, the uptake of 59Fe exceeded that of 125I. In dissociation studies the cellular 59Fe-activity was only partly dissociable during 16 h, whereas the 125I-activity could be released nearly completely. Disruption of the cells and studies on the cytosolic phase were performed employing gel-filtration and affinity chromatography. An appreciable amount of lactoferrin was found in the cytosolic phase. About 50% of the cytosolic 59Fe-activity was bound to ferritin. The results suggest that lactoferrin is internalized into monocytes and that iron is transferred to ferritin. These cellular events may contribute to an understanding of the accumulation of iron in the monocyte/macrophage system observed during inflammatory conditions.

Cells, Cultured↗
PubMed1987

Chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils.

A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revealed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.

Alkaline Phosphatase↗
PubMed1987

Beta 2-microglobulin in neutrophils: an intragranular protein.

The subcellular localization of beta2-microglobulin (beta 2m) in human neutrophils was determined by an enzyme-linked immunosorbent assay on subcellular fractions obtained by Percoll density gradient centrifugation of neutrophils disrupted by nitrogen cavitation. The neutrophils were found to contain 160 ng beta 2m/mg protein. Approximately two-thirds co-located with the markers for specific granules and was released from intact cells during degranulation, whereas one-third of the beta 2m was located together with markers of the plasma membrane. This fraction was not further enriched during degranulation. These results indicate that beta 2m cannot be universally used as a plasma membrane marker as hitherto assumed, but beta 2m may serve as an indicator of neutrophil degranulation.

Cell Compartmentation↗