scholarly journals Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules

1992 ◽  
Vol 284 (2) ◽  
pp. 513-520 ◽  
Author(s):  
S J Suchard ◽  
M J Burton ◽  
S J Stoehr
Keyword(s):  
Cytochalasin B ◽  
Anion Channel ◽  
Polymorphonuclear Leucocyte ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Surface Receptors ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Disulphonic Acid

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.

scholarly journals Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.

1990 ◽  
Vol 171 (4) ◽  
pp. 1155-1162 ◽  
Author(s):  
P A Detmers ◽  
S K Lo ◽  
E Olsen-Egbert ◽  
A Walz ◽  
M Baggiolini ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Binding Activity ◽  
Receptor Expression ◽  
Human Neutrophils ◽  
Adhesion Receptor ◽  
Biological Responses ◽  
Specific Granules ◽  
Inflammatory Stimuli ◽  
Adhesive Interactions

The cytokine NAP-1/IL-8 is produced by a variety of different cells in response to inflammatory stimuli and elicits several biological responses from PMN. Experiments presented here demonstrate that PMN exposed to NAP-1/IL-8 expressed increased amounts of CD11b/CD18, as well as CD11c/CD18 and CR1, on their cell surface, while expression of Fc gamma RIII and HLA-A,B,C remained essentially unchanged. Increased CD11b/CD18 and CD11c/CD18 appears to correspond with the release of specific granules by NAP-1/IL-8. NAP-1/IL-8 was also a potent stimulator of several of the binding activities of CD11b/CD18. Ligation of EC3bi by CD11b/CD18 was rapidly enhanced by NAP-1/IL-8, but phagocytosis of the ligated particles was not induced by the agonist. In addition, enhanced binding of EC3bi was observed in the absence of an increase in receptor expression as shown with PMN cytoplasts. NAP-1/IL-8 promoted additional adhesive interactions between CD11b/CD18 and the biosynthetic precursor of LPS, lipid IVa, fibrinogen, and endothelial cells, suggesting that NAP-1/IL-8 may promote leukocyte adhesion in vivo that could lead to recruitment of PMN to sites of tissue inflammation.

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

1991 ◽  
Vol 279 (3) ◽  
pp. 657-664 ◽  
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.

scholarly journals In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 242-250 ◽  
Author(s):  
J Calafat ◽  
R Goldschmeding ◽  
PL Ringeling ◽  
H Janssen ◽  
CE van der Schoot
Keyword(s):  
Immunoelectron Microscopy ◽  
Serine Proteases ◽  
Cytochalasin B ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Double Labeling ◽  
Specific Granules ◽  
Normal Human ◽  
Myeloid Leukocytes

Abstract Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA- antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA- positive patients and a mouse monoclonal antibody against the ANCA- antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti- myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B- treated neutrophils stimulated with N-formyl-methionyl-leucyl- phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.

scholarly journals The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes.

1989 ◽  
Vol 109 (6) ◽  
pp. 2771-2782 ◽  
Author(s):  
K A Joiner ◽  
T Ganz ◽  
J Albert ◽  
D Rotrosen
Keyword(s):  
Vitamin B12 ◽  
Salmonella Typhimurium ◽  
Human Serum ◽  
Human Neutrophils ◽  
Rabbit Igg ◽  
Receptor Interactions ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Normal Human ◽  
Specific Granule

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.

scholarly journals In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 242-250 ◽  
Author(s):  
J Calafat ◽  
R Goldschmeding ◽  
PL Ringeling ◽  
H Janssen ◽  
CE van der Schoot
Keyword(s):  
Immunoelectron Microscopy ◽  
Serine Proteases ◽  
Cytochalasin B ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Double Labeling ◽  
Specific Granules ◽  
Normal Human ◽  
Myeloid Leukocytes

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA- antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA- positive patients and a mouse monoclonal antibody against the ANCA- antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti- myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B- treated neutrophils stimulated with N-formyl-methionyl-leucyl- phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.

scholarly journals Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.

1983 ◽  
Vol 158 (4) ◽  
pp. 1160-1177 ◽  
Author(s):  
B N Cronstein ◽  
S B Kramer ◽  
G Weissmann ◽  
R Hirschhorn
Keyword(s):  
Superoxide Anion ◽  
Calcium Ionophore ◽  
Neutrophil Function ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Surface Receptors ◽  
Superoxide Anion Generation ◽  
Marked Enhancement ◽  
Nucleoside Uptake

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.

scholarly journals Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes.

1976 ◽  
Vol 68 (3) ◽  
pp. 781-787 ◽  
Author(s):  
S Hoffstein ◽  
R Soberman ◽  
I Goldstein ◽  
G Weissmann
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Microtubule Assembly ◽  
Human Neutrophils ◽  
Con A ◽  
Specific Granules ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.

scholarly journals Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.

1986 ◽  
Vol 102 (6) ◽  
pp. 2197-2204 ◽  
Author(s):  
P D Lew ◽  
A Monod ◽  
F A Waldvogel ◽  
B Dewald ◽  
M Baggiolini ◽  
...  
Keyword(s):  
Steady State ◽  
Cytochalasin B ◽  
Receptor Activation ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Secretory Vesicles ◽  
Cytosolic Free Calcium ◽  
Additional Signal ◽  
Calcium Indicator ◽  
Specific Granules

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

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.

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