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 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.

The Carriers of Native Vitamin B12 in Normal Human Serum

1977 ◽  
Vol 53 (5) ◽  
pp. 453-457 ◽  
Author(s):  
C. A. Hall
Keyword(s):  
Vitamin B12 ◽  
Human Serum ◽  
Isotope Dilution ◽  
Serum Vitamin ◽  
Total Serum ◽  
Plasma Vitamin ◽  
Isotope Dilution Assay ◽  
Dilution Assay ◽  
Normal Human ◽  
Transcobalamin Ii

1. After fractionation of the vitamin B12-binding proteins of ten normal sera the components containing transcobalamin II and R-type binders of vitamin B12 respectively were studied for endogenous vitamin B12 content by two distinct systems of vitamin B12 assay. 2. The measurements of total serum vitamin B12 by either bioassay with Euglena gracilis or a radioisotope dilution assay agreed closely. 3. The native vitamin B12 carried by transcobalamin II was higher as measured by bioassay than by isotope dilution assay. 4. The presence of the transcobalamin II fraction of human serum altered the key reaction between the binding reagent of the isotope dilution assay, so that this assay failed to measure vitamin B12 quantitatively. 5. Probably, the mean fraction of plasma vitamin B12 carried by transcobalamin II is in the range 20–30%.

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 Interaction of rheumatoid factor and Entamoeba histolytica

1989 ◽  
Vol 31 (4) ◽  
pp. 207-212
Author(s):  
P.G. Kremsner ◽  
W. Graninger
Keyword(s):  
Human Serum ◽  
Rheumatoid Factor ◽  
Entamoeba Histolytica ◽  
Normal Human Serum ◽  
Cytotoxicity Test ◽  
Marked Inhibition ◽  
Rabbit Igg ◽  
Lysis Rate ◽  
Normal Human

The amoebae's cytotoxicity test and the amoebae's lysis test were used to show possible interactions between rheumatoid factor (RF) and Entamoeba histolytica. Amoebae's cytotoxic activity (ACA) was inhibited by affinity chromatography purified antiamoebae rabbit IgG (RIgG). Enhanced inhibition could be demonstrated with RIgG plus RF. But the same marked inhibition of ACA could be seen when replacing RF by heat inactivated normal human serum as a control. About 50% amoebae's lysis occurred when amoebae were brought together with native normal human serum (NNHS) as a source of complement. Amoebae's lysis increased to 60% when incubated with NHS plus human antiamoebae antibodies. No further augmentation could be obtained by the addition of RF. Using RIgG instead of human antibodies the lysis rate did not increase. Incubation of amoebae, NNHS, RIgG and RF even reduced amoebae's lysis. RF neither has an effect on ACA nor on complement mediated AL in vitro.

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.

Silibinin (Legalon-70®) enhances the motility of human neutrophils immobilized by formyl-tripeptide, calcium ionophore, lymphokine and by normal human serum

1990 ◽  
Vol 29 (3-4) ◽  
pp. 239-246 ◽  
Author(s):  
L. Kalmár ◽  
J. Kádár ◽  
A. Somogyi ◽  
P. Gergely ◽  
G. Csomós ◽  
...  
Keyword(s):  
Human Serum ◽  
Calcium Ionophore ◽  
Normal Human Serum ◽  
Human Neutrophils ◽  
Normal Human

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.

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