Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj2990473 ◽

1994 ◽

Vol 299 (2) ◽

pp. 473-479 ◽

Cited By ~ 104

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3000325 ◽

1994 ◽

Vol 300 (2) ◽

pp. 325-330 ◽

Cited By ~ 28

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.

Download Full-text

Dual effects of guanosine 5′-[γ-thio]triphosphate on secretion by electroporated human neutrophils

Biochemical Journal ◽

10.1042/bj2790657 ◽

1991 ◽

Vol 279 (3) ◽

pp. 657-664 ◽

Cited By ~ 4

Author(s):

J E Smolen ◽

S J Stoehr ◽

B Kuczynski ◽

E K Koh ◽

G M Omann

Keyword(s):

G Proteins ◽

Cell Activation ◽

Adenine Nucleotides ◽

Human Neutrophils ◽

Inhibitory Effects ◽

Specific Granules ◽

Azurophil Granule ◽

Specific Granule ◽

Granule Secretion ◽

Granule Release

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Download Full-text

Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽

10.1182/blood.v83.6.1640.bloodjournal8361640 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1640-1649 ◽

Cited By ~ 2

Author(s):

L Kjeldsen ◽

H Sengelov ◽

K Lollike ◽

MH Nielsen ◽

N Borregaard

Keyword(s):

Plasma Membrane ◽

Qualitative Difference ◽

Human Neutrophils ◽

Immunogold Electron Microscopy ◽

Double Labeling ◽

Intact Cells ◽

Cytochrome B558 ◽

Isolation And Characterization ◽

Specific Granules

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.

Download Full-text

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 ◽

10.1182/blood.v83.3.808.808 ◽

1994 ◽

Vol 83 (3) ◽

pp. 808-815 ◽

Cited By ~ 108

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.

Download Full-text

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 ◽

10.1182/blood.v83.3.808.bloodjournal833808 ◽

1994 ◽

Vol 83 (3) ◽

pp. 808-815 ◽

Cited By ~ 1

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

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.97.1.52 ◽

1983 ◽

Vol 97 (1) ◽

pp. 52-61 ◽

Cited By ~ 601

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.

Download Full-text

Presence of Proteinase 3 in Secretory Vesicles: Evidence of a Novel, Highly Mobilizable Intracellular Pool Distinct From Azurophil Granules

Blood ◽

10.1182/blood.v94.7.2487.419k07_2487_2496 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2487-2496 ◽

Cited By ~ 120

Author(s):

Véronique Witko-Sarsat ◽

Elisabeth M. Cramer ◽

Corinne Hieblot ◽

Josette Guichard ◽

Patrick Nusbaum ◽

...

Keyword(s):

Plasma Membrane ◽

Serine Proteinase ◽

Antineutrophil Cytoplasmic Antibodies ◽

Human Neutrophils ◽

Secretory Vesicles ◽

Proteinase 3 ◽

Anca Associated Vasculitis ◽

Specific Granules ◽

Relevant Role ◽

Target Autoantigen

Proteinase 3 (PR3), which is also called myeloblastin, the target autoantigen for antineutrophil cytoplasmic antibodies (ANCA) in Wegener’s granulomatosis, is a serine proteinase stored in azurophil granules of human neutrophils. We have previously shown that, in contrast to elastase or myeloperoxidase, PR3 is also expressed at the plasma membrane of a subset of unactivated neutrophils and that a high proportion of neutrophils expressing membrane PR3 is a risk factor for vasculitis. The present study demonstrates that the association of PR3 with the plasma membrane is not an ionic interaction and seems to be covalent. Fractionation of neutrophils shows that, besides the azurophil granules, PR3 could be detected both in specific granules and in the plasma membrane-enriched fraction containing secretory vesicles, whereas elastase and myeloperoxidase were exclusively located in azurophil granules. Electron microscopy confirms that PR3 is present along with CR1 in secretory vesicles as well as in some specific granules. In neutrophils stimulated with an increasing dose of FMLP, membrane PR3 expression increased with the degranulation of secretory vesicles, followed by specific granules, and culminated after azurophil granules mobilization. The presence of a readily plasma membrane-mobilizable pool of PR3 contained in the secretory vesicles might play a relevant role in the pathophysiological mechanisms of ANCA-associated vasculitis.

Download Full-text