scholarly journals ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES

1966 ◽  
Vol 28 (2) ◽  
pp. 277-301 ◽  
Author(s):  
Dorothy Ford Bainton ◽  
Marilyn G. Farquhar
Keyword(s):  
Bone Marrow ◽  
Golgi Complex ◽  
Polymorphonuclear Leukocyte ◽  
Polymorphonuclear Leukocytes ◽  
Dense Core ◽  
Granule Formation ◽  
Specific Granules ◽  
Azurophil Granule ◽  
Rabbit Bone ◽  
Specific Granule

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO4. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (∼800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (∼500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.

scholarly journals Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.

1979 ◽  
Vol 27 (2) ◽  
pp. 665-675 ◽  
Author(s):  
D M Williams ◽  
R Gillett ◽  
J E Linder
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Electron Microscope ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Granule Formation ◽  
Specific Granules ◽  
Phosphatase Cytochemistry ◽  
Specific Granule ◽  
Rat Bone

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.

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

1968 ◽  
Vol 39 (2) ◽  
pp. 299-317 ◽  
Author(s):  
Dorothy Ford Bainton ◽  
Marilyn G. Farquhar
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Metal Salt ◽  
Histochemical Staining ◽  
Secretory Product ◽  
Enzyme Content ◽  
Specific Granules ◽  
Rabbit Bone ◽  
Alkaline Phosphate ◽  
Pmn Leukocyte

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.

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 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 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 STUDIES ON ISOLATED MEMBRANES OF AZUROPHIL AND SPECIFIC GRANULES FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

1972 ◽  
Vol 54 (1) ◽  
pp. 133-140 ◽  
Author(s):  
Ralph Nachman ◽  
James G. Hirsch ◽  
Marco Baggiolini
Keyword(s):  
Polymorphonuclear Leukocyte ◽  
Polymorphonuclear Leukocytes ◽  
Differential Centrifugation ◽  
Polyacrylamide Gels ◽  
Specific Granules ◽  
Ultrastructural Appearance ◽  
Protein Components

Membranes were prepared from rabbit polymorphonuclear leukocyte azurophil and specific granules separated by zonal differential centrifugation. The two types of granule membranes were quite similar in ultrastructural appearance, but they showed distinct differences in cholesterol-phospholipid ratios and in protein components demonstrable in polyacrylamide gels.

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.

Regulation of enzyme release from human polymorphonuclear leukocytes: further evidence for the independent regulation of granule subpopulations

1987 ◽  
Vol 65 (12) ◽  
pp. 1007-1015 ◽  
Author(s):  
Patricia Murphy ◽  
David A. Hart
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Second Messenger ◽  
Cytochalasin D ◽  
Enzyme Release ◽  
Specific Granules ◽  
Second Messenger Systems ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

Addition of 5–20 mM LiCl to purified human polymorphonuclear leukocytes led to the release of lysozyme, the specific granule constituent, but not the release of elastase which is in azurophilic granules. In contrast, 2.5–10 μg cytochalasin D/mL induced the release of both lysozyme and elastase. Addition of lipopolysaccharide to leukocytes did not induce enzyme release but primed cells for enhanced release induced by cytochalasin D. Lipopolysaccharide also primed cells for enhanced release of lysozyme by either N-formylmethionylleucylphenylalanine (fMLP) or Li+ but did not prime cells for elastase release by these stimuli. In contrast, fMLP + cytochalasin D interacted synergistically, leading to enhanced elastase release but not lysozyme release from the cells. Additional experiments with combinations of secretogogues and lipopolysaccharide yielded results consistent with the hypothesis that specific granules and subpopulations of azurophilic granules are under separate regulation and, thus, may be influenced by separate elements of intracellular second messenger systems.

scholarly journals Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate.

1975 ◽  
Vol 66 (3) ◽  
pp. 647-652 ◽  
Author(s):  
I M Goldstein ◽  
S T Hoffstein ◽  
G Weissmann
Keyword(s):  
Phorbol Myristate Acetate ◽  
Lysosomal Enzyme ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Enzyme Release ◽  
Myristate Acetate ◽  
Azurophil Granule ◽  
Cytoplasmic Microtubules ◽  
Specific Granule ◽  
Human Polymorphonuclear Leukocytes

PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a. PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli. Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a. In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells. Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells. Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.

scholarly journals CYTOPLASMIC GRANULE FORMATION IN MYELOCYTES

1966 ◽  
Vol 29 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Martha E. Fedorko ◽  
James G. Hirsch
Keyword(s):  
Amino Acids ◽  
Bone Marrow ◽  
Electron Microscope ◽  
Golgi Complex ◽  
Time Sequence ◽  
Granule Formation ◽  
Initial Exposure ◽  
Cytoplasmic Granules

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.

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