Isolation and characterization of gelatinase granules from human neutrophils

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.

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Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽

10.1182/blood.v83.6.1640.1640 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1640-1649 ◽

Cited By ~ 138

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.

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Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Blood ◽

10.1182/blood.v76.10.2105.bloodjournal76102105 ◽

1990 ◽

Vol 76 (10) ◽

pp. 2105-2116 ◽

Cited By ~ 1

Author(s):

LA Ginsel ◽

JJ Onderwater ◽

JA Fransen ◽

AJ Verhoeven ◽

D Roos

Keyword(s):

Plasma Membrane ◽

Human Peripheral Blood ◽

Polyclonal Antiserum ◽

Double Labeling ◽

Cytochrome B558 ◽

Inner Surface ◽

Specific Granules ◽

Membrane Bound ◽

P22 Phox ◽

Cytoplasmic Side

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.

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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 ◽

10.1182/blood.v82.10.3183.3183 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3183-3191 ◽

Cited By ~ 4

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.

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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 ◽

10.1182/blood.v82.10.3183.bloodjournal82103183 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3183-3191 ◽

Cited By ~ 83

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.

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Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Blood ◽

10.1182/blood.v76.10.2105.2105 ◽

1990 ◽

Vol 76 (10) ◽

pp. 2105-2116 ◽

Cited By ~ 30

Author(s):

LA Ginsel ◽

JJ Onderwater ◽

JA Fransen ◽

AJ Verhoeven ◽

D Roos

Keyword(s):

Plasma Membrane ◽

Human Peripheral Blood ◽

Polyclonal Antiserum ◽

Double Labeling ◽

Cytochrome B558 ◽

Inner Surface ◽

Specific Granules ◽

Membrane Bound ◽

P22 Phox ◽

Cytoplasmic Side

Abstract Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22- phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.

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Intracellular localization of glycosyl-phosphatidylinositol-anchored CD67 and FcRIII (CD16) in affected neutrophil granulocytes of patients with paroxysmal nocturnal hemoglobinuria

Blood ◽

10.1182/blood.v78.11.3030.bloodjournal78113030 ◽

1991 ◽

Vol 78 (11) ◽

pp. 3030-3036 ◽

Cited By ~ 1

Author(s):

CR Jost ◽

ML Gaillard ◽

JA Fransen ◽

MR Daha ◽

LA Ginsel

Keyword(s):

Flow Cytometry ◽

Plasma Membrane ◽

Immunoelectron Microscopy ◽

Golgi Complex ◽

Intracellular Localization ◽

Human Neutrophils ◽

Neutrophil Granulocytes ◽

Double Labeling ◽

Glycosyl Phosphatidylinositol ◽

Specific Granules

Immunoelectron microscopical studies performed in healthy human neutrophils showed the presence of glycosyl-phosphatidylinositol (GPI)- linked CD67 in granules. The use of immunogold double-labeling of CD67 and lactoferrin (LF; as marker for specific granules) or CD67 and myeloperoxidase (MPO; as marker for azurophilic granules) showed that CD67 occurred only in the specific granules. Furthermore, flow cytometry showed that CD67 has a low level of expression on the plasma membrane of these cells. In paroxsymal nocturnal hemoglobinuria (PNH)- affected neutrophils, CD67 was not detected in any intracellular compartment by immunoelectron microscopy, and flow cytometry showed no CD67 on the plasma membrane. In earlier studies, FcRIII was found on the plasma membrane, in electron-lucent vesicles, and in the Golgi complex of healthy neutrophils, and in the Golgi complex of some of the PNH-affected neutrophils. Here we have studied FcRIII in PNH-affected cells of three other patients and found, by immunoelectron microscopy, that the receptor can not be detected in these cells. However, flow cytometry showed that FcRIII was not completely absent on the plasma membrane of the affected cells, but that the level of expression on these cells was low. Thus, PNH patients can differ from one another with respect to the occurrence of affected neutrophils that have a detectable level of FcRIII in the Golgi complex. In summary, these findings show not only that the expression of the two GPI-linked proteins, CD67 and FcRIII, is markedly lower on the plasma membrane, but also that neither occurred in any of the intracellular compartments of affected neutrophils of the PNH patients examined in this study.

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Intracellular localization of glycosyl-phosphatidylinositol-anchored CD67 and FcRIII (CD16) in affected neutrophil granulocytes of patients with paroxysmal nocturnal hemoglobinuria

Blood ◽

10.1182/blood.v78.11.3030.3030 ◽

1991 ◽

Vol 78 (11) ◽

pp. 3030-3036 ◽

Cited By ~ 17

Author(s):

CR Jost ◽

ML Gaillard ◽

JA Fransen ◽

MR Daha ◽

LA Ginsel

Keyword(s):

Flow Cytometry ◽

Plasma Membrane ◽

Immunoelectron Microscopy ◽

Golgi Complex ◽

Intracellular Localization ◽

Human Neutrophils ◽

Neutrophil Granulocytes ◽

Double Labeling ◽

Glycosyl Phosphatidylinositol ◽

Specific Granules

Abstract Immunoelectron microscopical studies performed in healthy human neutrophils showed the presence of glycosyl-phosphatidylinositol (GPI)- linked CD67 in granules. The use of immunogold double-labeling of CD67 and lactoferrin (LF; as marker for specific granules) or CD67 and myeloperoxidase (MPO; as marker for azurophilic granules) showed that CD67 occurred only in the specific granules. Furthermore, flow cytometry showed that CD67 has a low level of expression on the plasma membrane of these cells. In paroxsymal nocturnal hemoglobinuria (PNH)- affected neutrophils, CD67 was not detected in any intracellular compartment by immunoelectron microscopy, and flow cytometry showed no CD67 on the plasma membrane. In earlier studies, FcRIII was found on the plasma membrane, in electron-lucent vesicles, and in the Golgi complex of healthy neutrophils, and in the Golgi complex of some of the PNH-affected neutrophils. Here we have studied FcRIII in PNH-affected cells of three other patients and found, by immunoelectron microscopy, that the receptor can not be detected in these cells. However, flow cytometry showed that FcRIII was not completely absent on the plasma membrane of the affected cells, but that the level of expression on these cells was low. Thus, PNH patients can differ from one another with respect to the occurrence of affected neutrophils that have a detectable level of FcRIII in the Golgi complex. In summary, these findings show not only that the expression of the two GPI-linked proteins, CD67 and FcRIII, is markedly lower on the plasma membrane, but also that neither occurred in any of the intracellular compartments of affected neutrophils of the PNH patients examined in this study.

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