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 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

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 Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 799-807 ◽  
Author(s):  
L Kjeldsen ◽  
DF Bainton ◽  
H Sengelov ◽  
N Borregaard
Keyword(s):  
Matrix Protein ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Distribution Profile ◽  
Double Labeling ◽  
Thin Sections ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Neutrophil Gelatinase ◽  
High Degree

Abstract Neutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

scholarly journals Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 799-807 ◽  
Author(s):  
L Kjeldsen ◽  
DF Bainton ◽  
H Sengelov ◽  
N Borregaard
Keyword(s):  
Matrix Protein ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Distribution Profile ◽  
Double Labeling ◽  
Thin Sections ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Neutrophil Gelatinase ◽  
High Degree

Neutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

scholarly journals Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1640-1649 ◽  
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.

scholarly journals Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1640-1649 ◽  
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

Abstract 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.

scholarly journals Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 423-432 ◽  
Author(s):  
E Cramer ◽  
KB Pryzwansky ◽  
JL Villeval ◽  
U Testa ◽  
J Breton-Gorius
Keyword(s):  
Human Peripheral Blood ◽  
Ultrastructural Localization ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Thin Sections ◽  
The Matrix ◽  
Blood Neutrophils ◽  
Microscopic Studies ◽  
Specific Localization ◽  
Monospecific Antibodies

Abstract Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

scholarly journals Platelet dense granule membranes contain both granulophysin and P- selectin (GMP-140)

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 143-152 ◽  
Author(s):  
SJ Israels ◽  
JM Gerrard ◽  
YV Jacques ◽  
A McNicol ◽  
B Cham ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Serotonin Release ◽  
Dense Granule ◽  
Double Labeling ◽  
Platelet Surface ◽  
Thin Sections ◽  
Dense Granules ◽  
Granule Membrane ◽  
Dense Granule Secretion

We recently reported the characterization of a platelet granule membrane protein of molecular weight (mol wt) 40,000 called granulophysin (Gerrard et al: Blood 77:101, 1991), identified by a monoclonal antibody (MoAb D545) raised to purified dense granule membranes. Using immunoelectron-microscopic techniques on frozen thin sections, this protein was localized in resting and thrombin-stimulated platelets. In resting platelets, labeled with antigranulophysin antibodies and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section. D545 also labeled dense granules in permeabilized whole platelets and isolated dense granule preparations examined by whole-mount techniques. Expression of granulophysin on the platelet surface paralleled dense granule secretion as measured by 14C-serotonin release under conditions in which lysosomal granule release, as measured by beta-glucuronidase secretion, was less than 5%. After thrombin stimulation, both the surface-connected canalicular system and the plasma membrane were labeled, demonstrating redistribution of granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the alpha-granule membrane protein, P-selectin, demonstrated labeling of both P-selectin and granulophysin on dense granule membranes. Distribution of both proteins on the plasma membrane after platelet stimulation was similar. The results demonstrate that granulophysin is localized to the dense granules of platelets and is redistributed to the plasma membrane after platelet activation.

scholarly journals Characterization of the SpaCBA Pilus Fibers in the Probiotic Lactobacillus rhamnosus GG

2012 ◽  
Vol 78 (7) ◽  
pp. 2337-2344 ◽  
Author(s):  
Justus Reunanen ◽  
Ingemar von Ossowski ◽  
Antoni P. A. Hendrickx ◽  
Airi Palva ◽  
Willem M. de Vos
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Immunogold Electron Microscopy ◽  
Probiotic Properties ◽  
Long Distance ◽  
Binding Ability ◽  
Intimate Contact ◽  
Double Labeling ◽  
Thin Sections ◽  
Content Type

ABSTRACTLactobacillus rhamnosusGG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown thatL. rhamnosusGG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A.106:17193–17198, 2009). These pili were found to be encoded by thespaCBAgene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to thespaCBAcluster, another putative pilus cluster,spaFED, was predicted from theL. rhamnosusGG genome sequence. Herein, we show that only SpaCBA pili are produced byL. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed forL. rhamnosusGG compared to that of nonpiliated lactobacilli.

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 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.

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