scholarly journals SEQUENTIAL DEGRANULATION OF THE TWO TYPES OF POLYMORPHONUCLEAR LEUKOCYTE GRANULES DURING PHAGOCYTOSIS OF MICROORGANISMS

1973 ◽  
Vol 58 (2) ◽  
pp. 249-264 ◽  
Author(s):  
Dorothy Ford Bainton
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocyte ◽  
Lysosomal Enzymes ◽  
Marker Enzyme ◽  
Optimal Conditions ◽  
Alkaline Ph ◽  
Enzyme Content ◽  
Specific Granules ◽  
Acid Range

The sequential discharge of neutrophilic polymorphonuclear leukocyte (PMN) granules—azurophils and specifics—was investigated by electron microscopy and cytochemistry. Thus the enzyme content of PMN phagocytic vacuoles was determined at brief intervals after phagocytosis of bacteria, utilizing peroxidase as a marker enzyme for azurophil granules, and alkaline phosphatase for specifics. At 30 s, approximately half the phagocytic vacuoles were reactive for alkaline phosphatase, whereas none contained peroxidase. Peroxidase-containing vacuoles were rarely seen at 1 min, but by 3 min, vacuoles containing both enzymes were consistently present. Alkaline phosphatase was found in both small and large vacuoles, whereas peroxidase was visible only in large ones. By 10 min, very big phagocytic vacuoles containing considerable amounts of reaction product for both enzymes were evident. These observations indicate that the two types of PMN granules discharge in a sequential manner, specific granules fusing with the vacuole before azurophils. In an earlier paper, we reported that the pH of phagocytic vacuoles drops to 6.5 within 3 min and to ∼4 within 7–15 min. Substances known to be present in specific granules (alkaline phosphatase, lysozyme, and lactoferrin) function best at neutral or alkaline pH, whereas most of those contained in azurophil granules (i.e., peroxidase and the lysosomal enzymes) have pH optima in the acid range. Hence the sequence of granule discharge roughly parallels the change in pH, thereby providing optimal conditions for coordinated activity of granule contents.

scholarly journals DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES

1968 ◽  
Vol 39 (2) ◽  
pp. 286-298 ◽  
Author(s):  
Dorothy Ford Bainton ◽  
Marilyn G. Farquhar
Keyword(s):  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocytes ◽  
Basic Protein ◽  
Developmental Stages ◽  
Hydrolytic Enzymes ◽  
Enzyme Content ◽  
Specific Granules ◽  
Rabbit Bone ◽  
Thiolacetic Acid ◽  
Enzyme Alkaline Phosphatase

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, ß-galactosidase, ß-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.

scholarly journals CYTOCHEMISTRY AND ELECTRON MICROSCOPY

1963 ◽  
Vol 17 (1) ◽  
pp. 19-58 ◽  
Author(s):  
David D. Sabatini ◽  
Klaus Bensch ◽  
Russell J. Barrnett
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Fine Structure ◽  
Acid Phosphatase ◽  
Adenosine Triphosphatase ◽  
Osmium Tetroxide ◽  
Enzyme Histochemistry ◽  
Succinic Dehydrogenase ◽  
Optimal Conditions ◽  
Fixation Procedure

The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.

Azurophil and specific granules of blood neutrophils in chronic myelogenous leukemia: an ultrastructural and cytochemical analysis

Blood ◽  
1975 ◽  
Vol 45 (4) ◽  
pp. 469-482
Author(s):  
JL Ullyot ◽  
DF Bainton
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Terminal Phase ◽  
Leukocyte Alkaline Phosphatase ◽  
Specific Granules ◽  
Blood Neutrophils ◽  
Specific Granule

That most patients with chronic myelogenous leukemia (CML) have either very low levels or no leukocyte alkaline phosphatase activity (LAP) is an established fact. In view of our new findings7 that normal mature human polymorphonuclear leukocytes (PMN) contain two types of granules, azurophils (1/3) and specifics (2/3), and that alkaline phosphatase is present only in specific granules, we undertook the present studies to determine whether these neoplastic PMN lack a specific granule population or simply lack the enzyme. The cellular buffy coats of five patients with CML (Ph1 plus, LAP minus) were fixed in glutaraldehyde, incubated for peroxidase to identify the azurophil population, and examined by electron microscopy. It was found that the specific granule population was present in all mature PMN. Counts of both azurophil and specific granules per cell were slightly lower than normal but were within an 80%-90% overlap of the normal range. We therefore conclude that the low level of LAP in patients with CML reflects a deficiency of the enzyme rather than a missing granule population. Although the mature PMN appeared relatively normal (with few exceptions), circulating myeloblasts and promyelocytes revealed several abnormalities, the most notable being the presence of large bundles of cytoplasmic microfilaments. The blood of two patients in the terminal phase of disease was reexamined. Most of their cells were immature, with aberrations similar to those in myeloblasts and promyelocytes in the chronic phase of the disorder. In addition, however, we discovered three adnormal populations of mature PMN: (1) PMN containing both populations of granules but lacking peroxidase, (2) PMN lacking specific granules, and (3) PMN lacking azurophil granules. Our findings emphasize the value of electron microscopy and cytochemistry in detecting abnormalities of maturation in the cytoplasm of leukemic PMN.

scholarly journals Azurophil and Specific Granules of Blood Neutrophils in Chronic Myelogenous Leukemia: An Ultrastructural and Cytochemical Analysis

Blood ◽  
1974 ◽  
Vol 44 (4) ◽  
pp. 469-482 ◽  
Author(s):  
Joan L. Ullyot ◽  
Dorothy F. Bainton
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Terminal Phase ◽  
Leukocyte Alkaline Phosphatase ◽  
Specific Granules ◽  
Blood Neutrophils ◽  
Specific Granule

Abstract That most patients with chronic myelogenous leukemia (CML) have either very low levels or no leukocyte alkaline phosphatase activity (LAP) is an established fact. In view of our new findings7 that normal mature human polymorphonuclear leukocytes (PMN) contain two types of granules, azurophils (1/3) and specifics (2/3), and that alkaline phosphatase is present only in specific granules, we undertook the present studies to determine whether these neoplastic PMN lack a specific granule population or simply lack the enzyme. The cellular buffy coats of five patients with CML (Ph1+, LAP-) were fixed in glutaraldehyde, incubated for peroxidase to identify the azurophil population, and examined by electron microscopy. It was found that the specific granule population was present in all mature PMN. Counts of both azurophil and specific granules per cell were slightly lower than normal but were within an 80%-90% overlap of the normal range. We therefore conclude that the low level of LAP in patients with CML reflects a deficiency of the enzyme rather than a missing granule population. Although the mature PMN appeared relatively normal (with few exceptions), circulating myeloblasts and promyelocytes revealed several abnormalities, the most notable being the presence of large bundles of cytoplasmic microfilaments. The blood of two patients in the terminal phase of disease was reexamined. Most of their cells were immature, with aberrations similar to those in myeloblasts and promyelocytes in the chronic phase of the disorder. In addition, however, we discovered three adnormal populations of mature PMN: (1) PMN containing both populations of granules but lacking peroxidase, (2) PMN lacking specific granules, and (3) PMN lacking azurophil granules. Our findings emphasize the value of electron microscopy and cytochemistry in detecting abnormalities of maturation in the cytoplasm of leukemic PMN.

Azurophil and specific granules of blood neutrophils in chronic myelogenous leukemia: an ultrastructural and cytochemical analysis

Blood ◽  
1975 ◽  
Vol 45 (4) ◽  
pp. 469-482
Author(s):  
JL Ullyot ◽  
DF Bainton
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Terminal Phase ◽  
Leukocyte Alkaline Phosphatase ◽  
Specific Granules ◽  
Blood Neutrophils ◽  
Specific Granule

Abstract That most patients with chronic myelogenous leukemia (CML) have either very low levels or no leukocyte alkaline phosphatase activity (LAP) is an established fact. In view of our new findings7 that normal mature human polymorphonuclear leukocytes (PMN) contain two types of granules, azurophils (1/3) and specifics (2/3), and that alkaline phosphatase is present only in specific granules, we undertook the present studies to determine whether these neoplastic PMN lack a specific granule population or simply lack the enzyme. The cellular buffy coats of five patients with CML (Ph1 plus, LAP minus) were fixed in glutaraldehyde, incubated for peroxidase to identify the azurophil population, and examined by electron microscopy. It was found that the specific granule population was present in all mature PMN. Counts of both azurophil and specific granules per cell were slightly lower than normal but were within an 80%-90% overlap of the normal range. We therefore conclude that the low level of LAP in patients with CML reflects a deficiency of the enzyme rather than a missing granule population. Although the mature PMN appeared relatively normal (with few exceptions), circulating myeloblasts and promyelocytes revealed several abnormalities, the most notable being the presence of large bundles of cytoplasmic microfilaments. The blood of two patients in the terminal phase of disease was reexamined. Most of their cells were immature, with aberrations similar to those in myeloblasts and promyelocytes in the chronic phase of the disorder. In addition, however, we discovered three adnormal populations of mature PMN: (1) PMN containing both populations of granules but lacking peroxidase, (2) PMN lacking specific granules, and (3) PMN lacking azurophil granules. Our findings emphasize the value of electron microscopy and cytochemistry in detecting abnormalities of maturation in the cytoplasm of leukemic PMN.

scholarly journals LYSOSOMAL AND ULTRASTRUCTURAL CHANGES IN HUMAN "TOXIC" NEUTROPHILS DURING BACTERIAL INFECTION

1969 ◽  
Vol 129 (2) ◽  
pp. 267-293 ◽  
Author(s):  
Charles E. McCall ◽  
Isao Katayama ◽  
Ramzi S. Cotran ◽  
Maxwell Finland
Keyword(s):  
Electron Microscopy ◽  
Alkaline Phosphatase ◽  
Bacterial Infection ◽  
Bacterial Infections ◽  
Neutral Red ◽  
Ultrastructural Changes ◽  
Staining Reaction ◽  
Total Acid ◽  
Dense Granules ◽  
Specific Granules

"Toxic" neutrophils from humans with severe bacterial infections, identified by the presence of Döhle bodies, "toxic" granules, and vacuoles were shown to differ from normal neutrophils both in ultrastructure and in lysosome activity. Döhle bodies were identified as lamellar aggregates of rough endoplasmic reticulum. Toxic granules corresponded to the azurophilic granules usually identified by Romanowsky stains only in neutrophil precursors. By electron microscopy such granules were large, electron-dense, and peroxidase positive; they could usually be distinguished from the smaller, less dense, "specific" granules also present in control neutrophils, but in the latter they became visible by light microscopy only after prolonged staining or following fixation with glutaraldehyde. These observations suggest that toxic granules represent an abnormal staining reaction of the large dense granules in the toxic cells, and not phagocytized material, newly formed abnormal granules or autophagic bodies. Alkaline phosphatase activity was significantly greater in toxic neutrophils than in normal ones; 80% of the activity of both was located in the lysosome fraction. Beta glucuronidase was normal. Total acid phosphatase was normal, but the percentage located in the nonlysosome fraction of toxic neutrophils was increased, suggesting that lysosomes were "labilized." Formation of neutral red vacuoles in supravitally stained preparations, an index of lysosome activity, occurred more rapidly in toxic neutrophils. This reaction paralleled degranulation and the formation of clear vacuoles in unstained wet mounts and could be blocked by colchicine, a lysosome stabilizer, or enhanced by procedures which activate lysosomes. "Autophagic" vacuoles were observed by electron microscopy in some toxic neutrophils. These observations are discussed in relation to the concept that the "toxic" neutrophils in severe bacterial infection reflect cellular immaturity and/or stimulation or degeneration.

scholarly journals ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES

1973 ◽  
Vol 59 (3) ◽  
pp. 696-707 ◽  
Author(s):  
Ursula Bretz ◽  
Marco Baggiolini
Keyword(s):  
Alkaline Phosphatase ◽  
Polymorphonuclear Leukocyte ◽  
Sodium Sulfate ◽  
Polymorphonuclear Leukocytes ◽  
Sodium Deoxycholate ◽  
Soluble Form ◽  
Hexadecyltrimethylammonium Bromide ◽  
Specific Granules ◽  
Granule Membrane ◽  
Triton X

The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane.

scholarly journals The cobalt(II)-alkaline phosphatase system at alkaline pH.

1988 ◽  
Vol 263 (23) ◽  
pp. 11263-11268
Author(s):  
L Banci ◽  
I Bertini ◽  
C Luchinat ◽  
M S Viezzoli ◽  
Y J Wang
Keyword(s):  
Alkaline Phosphatase ◽  
Alkaline Ph ◽  
Phosphatase System

scholarly journals Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

1975 ◽  
Vol 23 (5) ◽  
pp. 342-347 ◽  
Author(s):  
A Linde ◽  
B C Magnusson
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Adenosine Triphosphatase ◽  
Ruthenium Red ◽  
Inorganic Pyrophosphatase ◽  
Alkaline Ph ◽  
Hard Tissue ◽  
Assay Condition ◽  
Phosphatase Inhibitors ◽  
Inhibition Studies

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.

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