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

1995 ◽  
Vol 311 (2) ◽  
pp. 667-674 ◽  
Author(s):  
C Dahlgren ◽  
S R Carlsson ◽  
A Karlsson ◽  
H Lundqvist ◽  
C Sjölin
Keyword(s):  
Membrane Fraction ◽  
Plasma Membranes ◽  
Subcellular Fractionation ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Secretory Vesicles ◽  
Membrane Glycoproteins ◽  
Specific Granules ◽  
Protein Group

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.

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 Calcium-induced translocation of annexins to subcellular organelles of human neutrophils

1994 ◽  
Vol 300 (2) ◽  
pp. 325-330 ◽  
Author(s):  
C Sjölin ◽  
O Stendahl ◽  
C Dahlgren
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Consensus Sequence ◽  
Annexin Ii ◽  
Secretory Process ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Annexin I ◽  
Specific Granules ◽  
Annexin Iv

The annexins are Ca(2+)-regulated, phospholipid-binding proteins which have been suggested to take part in cellular events such as exocytosis. The subcellular localization of annexins in human neutrophils was determined using monoclonal antibodies against annexins I, II, IV and VI and a polyclonal peptide antiserum against an annexin consensus sequence. Several annexins were translocated to the light membrane fraction enriched in plasma membranes and secretory vesicles. Annexins were associated also with the azurophil and specific granules. Whereas annexins I, IV and VI and one unidentified 35 kDa protein translocated to each of the isolated organelles, annexin II, a 66 kDa annexin IV-like protein, and a 38 kDa annexin I-like protein exhibited organelle-related differences in their association with membranes. The 38 kDa annexin associated only with specific granules and the secretory vesicles/plasma membrane but not with azurophil granules. Annexin II and the 66 kDa annexin IV-like protein associated with each of the neutrophil organelles, but the binding to specific granules and secretory vesicles/plasma membrane showed a Ca(2+)-dependency different from that of azurophil granules. This observation suggests that these proteins may contribute to the secretory process in neutrophils.

scholarly journals Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.

1983 ◽  
Vol 97 (1) ◽  
pp. 52-61 ◽  
Author(s):  
N Borregaard ◽  
J M Heiple ◽  
E R Simons ◽  
R A Clark
Keyword(s):  
Plasma Membrane ◽  
Human Neutrophil ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Complete Separation ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Percoll Density Gradient Centrifugation ◽  
Specific Granules

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.

scholarly journals Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

1996 ◽  
Vol 314 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R. Alexander BLACKWOOD ◽  
James E. SMOLEN ◽  
Ronald J. HESSLER ◽  
Donna M. HARSH ◽  
Amy TRANSUE
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Phospholipid Vesicle ◽  
Human Neutrophils ◽  
Plasma Membrane Vesicles ◽  
Fluorescent Marker ◽  
Whole Cells ◽  
Specific Granules ◽  
Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

scholarly journals Comparison of Proteins Expressed on Secretory Vesicle Membranes and Plasma Membranes of Human Neutrophils

2008 ◽  
Vol 180 (8) ◽  
pp. 5575-5581 ◽  
Author(s):  
Silvia M. Uriarte ◽  
David W. Powell ◽  
Gregory C. Luerman ◽  
Michael L. Merchant ◽  
Timothy D. Cummins ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Vesicle Membranes

scholarly journals The Human Antibacterial Cathelicidin, hCAP-18, Is Synthesized in Myelocytes and Metamyelocytes and Localized to Specific Granules in Neutrophils

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2796-2803 ◽  
Author(s):  
Ole Sørensen ◽  
Kristina Arnljots ◽  
Jack B. Cowland ◽  
Dorothy F. Bainton ◽  
Niels Borregaard
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Subcellular Fractionation ◽  
Human Neutrophils ◽  
Major Protein ◽  
Equimolar Ratio ◽  
Binding Domains ◽  
Specific Granules ◽  
Immuno Electron Microscopy

hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.

The Role of the Small GTPase Rab27a in the Regulated Secretion of Granulocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3080-3080
Author(s):  
Sergio D. Catz ◽  
Daniela B. Munafo ◽  
Beverly A. Ellis ◽  
Jennifer L. Johnson
Keyword(s):  
Membrane Fraction ◽  
Small Gtpase ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Cell Fractionation ◽  
Immunofluorescence Analysis ◽  
Regulated Secretion ◽  
Immuno Electron Microscopy ◽  
Dmso Treatment

Abstract Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

A1AT In Human Neutrophil Granulocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3784-3784
Author(s):  
Stine Novrup Clemmensen ◽  
Lars C. Jacobsen ◽  
Sara Roervig ◽  
Bent Adel Hansen ◽  
Martin Iversen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Mrna Level ◽  
Cathepsin G ◽  
Neutrophil Granulocytes ◽  
Secretory Vesicles ◽  
Functional Abnormality ◽  
Structural Abnormalities ◽  
Specific Granules ◽  
A1at Deficiency

Abstract Abstract 3784 Alpha-1-antitrypsin (A1AT) is an important inhibitor of the neutrophil serine proteases elastase, cathepsin G, and proteinase 3. A1AT is produced mainly by the liver and secreted to plasma. A1AT deficiency caused by the PiZZ mutation in the A1AT gene leads to accumulation of mutated A1AT in the liver which may induce liver cell necrosis and necessitate liver transplantation. In a recently performed profiling of mRNA expression during terminal neutrophil differentiation in the bone marrow, we found that A1AT mRNA increases from the promyelocyte stage and up, indicating that A1AT is a constituent of all neutrophil granules. We examined the localization and production of A1AT in healthy donor neutrophils and investigated whether the structure or function of neutrophils is affected in individuals with A1AT deficiency. RT- PCR for A1AT performed on neutrophil precursors isolated from normal human bone marrow showed that the mRNA level is highly upregulated as the cells mature in the bone marrow and even increases further as the cells enter the blood stream. Biosynthesis studies revealed that A1AT is produced by all stages of neutrophil maturation in the bone marrow and is efficiently retained in the cells as judged by pulse chase studies. Neutrophils from circulating blood also produce A1AT but this is not retained in the cells. Stimulation of neutrophils from peripheral blood with G-CSF during 24 hours resulted in a 20 fold increase in A1AT biosynthesis which was largely released to the medium. Subcellullar fractionation of blood neutrophils on a 4-layer Percoll density gradient revealed 3 forms of A1AT. A doublet band at 37 and 44 kD both with immunoreactivity against A1AT was observed in fractions corresponding to azurophil granules (cf biosynthesis of this form in promyelocytes). A band with mw of 52 kD, corresponding to the form present in blood plasma, was observed in fractions that contain NGAL, a marker of specific granules and in fractions that contain gelatinase. The 52 kD band was also observed in fractions containing albumin as expected, since secretory vesicles contain plasma proteins. The localization of A1at in neutrophil granules was further confirmed by exocytosis studies. Neutrophils were stimulated with PMA which mobilizes secretory vesicles and gelatinase granules efficiently and approximately 50% of specific granules. Only the 52 mw form of A1AT was released from cells during stimulation while none of the 37/44 double band was released. This is in agreement with localization of this double band in azurophil granules and with localization of the 52 kD form in specific granules, gelatinase granules and secretory vesicles. In addition, a high molecular weight form of A1AT was observed at 76 kD corresponding to the mw of A1AT complexed with neutrophil elastase. We isolated neutrophils from patients with the ZZ genotype of A1AT deficiency which had either been liver transplanted or lung transplanted. The neutrophils were examined by electron microscopy for detection of structural abnormalities and by exocytosis studies for detection of functional abnormalities. Electron micrographs did not reveal any abnormality in neutrophil structure and in none of the neutrophils examined (from 6 patients) did we observe abnormal granules akin to the intracellular accumulation of A1AT in liver cells from patients. We did, however, observe reduction in the total intracellular amount of A1AT in neutrophils from patients that had been lung transplanted but not in neutrophils from liver transplanted patients. This most likely reflects that secretory vesicles of neutrophils from lung transplanted will not contain A1AT as this is still severely deficient in plasma from lung transplanted patients, while liver transplanted patients will have normal levels of A1AT in plasma and hence take up normal amounts into their secretory vesicles. Release of granule proteins in response to stimulation by fMLP or PMA did not reveal any functional abnormality in neutrophils from A1AT deficient patients. Based on these studies we conclude that the A1AT deficiency does not inflict functional or structural abnormalities on neutrophils, and suggest that A1AT generated and released from neutrophils may contribute to anti-protease defense in tissues. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of a distinct gelatinase- containing granule subset by combined immunocytochemistry and subcellular fractionation

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3183-3191 ◽  
Author(s):  
L Kjeldsen ◽  
DF Bainton ◽  
H Sengelov ◽  
N Borregaard
Keyword(s):  
Ultrastructural Localization ◽  
Subcellular Fractionation ◽  
Free Form ◽  
Human Neutrophils ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
Thin Sections ◽  
Dense Granules ◽  
Specific Granules ◽  
Neutrophil Gelatinase

Abstract The existence of separate gelatinase granules in human neutrophils has been a matter of debate in recent years. We have demonstrated that the 135-kD form of neutrophil gelatinase is a complex of 92-kD gelatinase and a novel 25-kD protein termed neutrophil gelatinase-associated lipocalin (NGAL) that, in addition to being complexed with part of the gelatinase, is localized in free form in peroxidase-negative specific granules. Because this association was not appreciated in earlier studies, we decided to reassess the ultrastructural localization of gelatinase using specific antibodies without immunoreactivity towards NGAL. Double-labeling immunogold electron microscopy was performed on frozen thin sections of human neutrophils. Twenty-four percent of all peroxidase-negative granules were labeled with antigelatinase antibody, but not with antilactoferrin antibody. These granules are defined as gelatinase granules. Sixteen percent reacted with antilactoferrin antibody but not with antigelatinase antibody. The rest (60%) reacted with both antibodies. All granules labeling for lactoferrin are defined as specific granules. Gelatinase granules were observed as round and oval forms of considerably smaller size than specific granules, and were less electron dense. Isolated granules obtained by subcellular fractionation were also examined by immunoelectron microscopy. This demonstrated that peroxidase-negative granules comprise a continuum from the most dense granules that contain lactoferrin but no gelatinase to the lightest that contain gelatinase but no lactoferrin. Thus, gelatinase granules do exist as a subpopulation of peroxidase-negative granules and may allow for exocytosis of gelatinase during neutrophil diapedesis without substantial mobilization of other peroxidase- negative granules, ie, specific granules.

scholarly journals The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 808-815 ◽  
Author(s):  
T Plesner ◽  
M Ploug ◽  
V Ellis ◽  
E Ronne ◽  
G Hoyer-Hansen ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Plasminogen Activator ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Plasminogen Activation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Granules ◽  
Stimulation Of

Abstract The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface-bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor-alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.

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