Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators

1994 ◽  
Vol 56 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Niels Borregaard ◽  
Lars Kieldsen ◽  
Henrik Sengelo ◽  
Michael S. Diamond ◽  
Timothy A. Springer ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Subcellular Localization ◽  
Inflammatory Mediators ◽  
Surface Expression ◽  
Human Neutrophils

scholarly journals Regulation of CD18 expression in human neutrophils as related to shape changes

1993 ◽  
Vol 106 (2) ◽  
pp. 493-501
Author(s):  
A. Volz
Keyword(s):  
Phorbol Myristate Acetate ◽  
Surface Expression ◽  
Cytochalasin D ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Myristate Acetate ◽  
Surface Distribution ◽  
Quantitative Expression ◽  
High Concentrations ◽  
Shape Changes

The study analyses the distribution and quantitative expression of surface CD18 of neutrophils exposed to distinct stimuli that produce different types of continuous shape changes, including types that are associated with locomotion and others that are not. The chemotactic peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanine, colchicine and nocodazole were used to induce a polarized locomotor morphology, phorbol myristate acetate, 1,2-dioctanoylglycerol and 1-oleoyl-2-acetyl-glycerol to induce non-polar motile cells ruffling all over the surface and 2H2O to induce non-polar cells performing circus movements as have been previously described. Except for colchicine and nocodazole, these stimuli increased surface expression of CD18. Thus, stimulated shape changes are frequently, though not always, associated with increased surface expression of CD18. High concentrations (10(−7) to 10(−5) M) of phorbol myristate acetate but not of chemotactic peptide induced down-regulation of surface CD18. Cytochalasin D (10(−4) M) stimulated CD18 expression even though it inhibited shape changes. The surface distribution of CD18 determined by light microscopy was uniform in unstimulated cells or in various forms of stimulation except for cells treated with 10(−5) M cytochalasin D. Cytochalasin D (10(−5) M) produced CD18 accumulation at the pole opposite the F-actin cap. Experiments with colchicine, nocodazole, 2H2O and cytochalasin D suggest that microtubules as well as microfilaments modulate surface expression of CD18. The results suggest that protein kinase C and phosphatases play a role in regulating surface expression of CD18 in neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface.

1993 ◽  
Vol 41 (9) ◽  
pp. 1367-1372 ◽  
Author(s):  
T J Cain ◽  
Y Liu ◽  
T Kobayashi ◽  
J M Robinson
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Human Neutrophils ◽  
Extracellular Medium ◽  
Isolation And Purification ◽  
Glycosyl Phosphatidylinositol ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Pre Treatment ◽  
Rapid Purification

Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.

Identification of intracellular sites of superoxide production in stimulated neutrophils

1998 ◽  
Vol 111 (1) ◽  
pp. 81-91 ◽  
Author(s):  
T. Kobayashi ◽  
J.M. Robinson ◽  
H. Seguchi
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Time Course ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Morphological Evidence ◽  
Marker Enzyme ◽  
Intracellular Compartments

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.

Inhibition of human neutrophil beta2-integrin-dependent adherence by hyperbaric O2

1997 ◽  
Vol 272 (3) ◽  
pp. C770-C777 ◽  
Author(s):  
S. R. Thom ◽  
I. Mendiguren ◽  
K. Hardy ◽  
T. Bolotin ◽  
D. Fisher ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Human Neutrophil ◽  
Animal Studies ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Neutrophils ◽  
Membrane Guanylate Cyclase ◽  
Binding Studies ◽  
Beta2 Integrins ◽  
Cell Fractions

Animal and clinical investigations have reported that exposure to hyperbaric O(2) improved the outcome of some reperfusion injuries. Animal studies have suggested that this may be due to an inhibition of leukocyte adherence to injured endothelium. This investigation tested the hypothesis that exposure to hyperbaric O(2) would inhibit beta2-integrin-dependent adherence of human neutrophils. Subjects were exposed to O(2) at partial pressures of up to 3 atmospheres absolute (ATA; 1 ATA = 0.1 MPa) for 45 min, and neutrophil binding to nylon columns and to fibrinogen-coated surfaces was measured. Exposure to O(2) at 2.8 or 3.0 ATA inhibited beta2-integrin-dependent neutrophil adherence but had no effect on the cell-surface expression of beta2-integrins, respiratory burst in response to phorbol ester, or non-beta2-integrin-dependent adherence to plastic plates coated with a fibronectin-like protein. beta2-Integrin adherence was restored by incubating blood with 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and hyperbaric O(2) inhibited synthesis of cGMP by neutrophils stimulated with N-formyl-Met-Leu-Phe (FMLP). In studies of cell fractions, the activity of membrane guanylate cyclase was found to be increased by incubation with FMLP as well as by atrial natriuretic peptide (ANP) plus ATP. Hyperbaric O(2) had no effect on the basal activity of soluble or membrane-bound guanylate cyclase. However, hyperbaric O(2) inhibited the function of both the extracellular binding domain of membrane guanylate cyclase as well as intracellular catalytic activity. There are approximately 7,300 membrane guanylate cyclase molecules per cell, based on binding studies with ANP, with a dissociation constant of approximately 450 pM. Hyperbaric O(2) inhibits the function of human neutrophil beta2-integrins by a process linked to impaired synthesis of cGMP. a

scholarly journals Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

1994 ◽  
Vol 299 (2) ◽  
pp. 473-479 ◽  
Author(s):  
H Sengeløv ◽  
F Boulay ◽  
L Kjeldsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Beta Band ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

scholarly journals The Thyroid Hormone Inactivating Enzyme Type 3 Deiodinase is Present in Bactericidal Granules and the Cytoplasm of Human Neutrophils

Endocrinology ◽  
2016 ◽  
Vol 157 (8) ◽  
pp. 3293-3305 ◽  
Author(s):  
Anne H. van der Spek ◽  
Flavia F. Bloise ◽  
Wikky Tigchelaar ◽  
Monica Dentice ◽  
Domenico Salvatore ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Subcellular Localization ◽  
Subcellular Location ◽  
Essential Elements ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Transcriptional Level ◽  
Bacterial Killing ◽  
Effector Cells ◽  
Type 3

Neutrophils are important effector cells of the innate immune system. Thyroid hormone (TH) is thought to play an important role in their function. Intracellular TH levels are regulated by the deiodinating enzymes. The TH-inactivating type 3 deiodinase (D3) is expressed in infiltrating murine neutrophils, and D3 knockout mice show impaired bacterial killing upon infection. This suggests that D3 plays an important role in the bacterial killing capacity of neutrophils. The mechanism behind this effect is unknown. We aimed to assess the presence of D3 in human neutrophils, and determine its subcellular localization using confocal and electron microscopy, because this could give important clues about its function in these cells. D3 appeared to be present in the cytoplasm and in myeloperoxidase containing azurophilic granules and as well as lactoferrin containing specific granules within human neutrophils. This subcellular localization did not change upon activation of the cells. D3 is observed intracellularly during neutrophil extracellular trap formation, followed by a reduction of D3 staining after release of the neutrophil extracellular traps into the extracellular space. At the transcriptional level, human neutrophils expressed additional essential elements of TH metabolism, including TH transporters and TH receptors. Here, we demonstrate the presence and subcellular location of D3 in human neutrophils for the first time and propose a model, in which D3 plays a role in the bacterial killing capacity of neutrophils either through generation of iodide for the myeloperoxidase system or through modulation of intracellular TH bioavailability.

CD39/NTPDase1 Variants Identified in Human Neutrophils Regulate Antithrombotic Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3088-3088 ◽  
Author(s):  
Kim E. Olson ◽  
Dianne Pulte ◽  
Marinus Johan Broekman ◽  
Ashley E. Olson ◽  
Joan Drosopoulos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Western Blot ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Neutrophils ◽  
Platelet Activity ◽  
Total Rna ◽  
Cos Cells

Abstract Blood-borne cellular elements expressing ectonucleotidase activity have been shown to regulate platelet activation and recruitment in response to agonists. In particular, exposure of a platelet releasate to isolated neutrophils (PMN) results in loss of its platelet activating activity in a subsequent assay (Valles et al, J Clin Invest1993, 92:1357–1365). Whereas expression of CD39 on vascular endothelial cells has been well characterized, expression on leukocytes has been less well studied. Freshly prepared lymphocyte and PMN cell populations were evaluated for both cell surface expression of CD39 and ectonucleotidase activity. FACS analysis showed that 98% of PMN were positive for CD39 compared to only 20% of lymphocytes. In addition, neutrophils stained more intensely, indicating the presence of a higher quantity of cell surface-expressed CD39. Interestingly, neutrophils exhibited only 1/3 of the ATPase and 1/2 of the ADPase activities of the same number of lymphocytes, although the latter are thought to have greater antithrombotic capacity. RT-PCR products from total RNA isolated from lymphocytes and PMN were sequenced. This revealed alternately spliced CD39 mRNA species present in PMN at levels equal to that of CD39 mRNA. In contrast, lymphocytes, which showed much higher levels of CD39 mRNA, expressed these variants at much lower levels. RACE analyses of cDNAs generated from total RNA demonstrated two CD39 gene-derived mRNAs. Each was comprised of an alternate 3′ segment lacking the C-terminal transmembrane domain, and distinguished by an internal deletion. Myc- and Flag-tagged constructs expressed in COS cells resulted in cell surface expression of the respectively tagged variants (immunocytochemistry, western blot analyses of plasma membrane preparations). Membrane preparations assayed for enzyme activity revealed no apyrase activity for either molecule expressed alone or together. Co-transfection of CD39 with equal amounts of either construct singly or in combination resulted in a 30-50% decrease in ATPase activity compared to CD39 alone. Similarly, CD39 co-expressed with either construct alone lost 75–90% of its ADPase activity. Unexpectedly, co-transfection of CD39 with both variants together resulted in a 20–40% increase in ADPase activity. Glutaraldehyde cross-linking of membrane preparations from triply transfected COS cells followed by immunoprecipitation and western blot analyses demonstrated the presence of all three species in higher order complexes. Thus, both variants can simultaneously associate with CD39, generating hetero-multimers with altered substrate preference and catalytic efficiency compared to CD39 tetramers. These observations add to our understanding of the regulation of ectonucleotidase activity at the cell surface. The balanced expression of CD39 and its two identified variants may underlie the anti-platelet activity of neutrophils previously reported. The finding that association of CD39 with either construct alone results in near complete loss of ADPase activity with only partial diminution of ATPase activity suggests a possible etiology for a pro-thrombotic phenotype.

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