Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes

1975 ◽  
Vol 17 (1) ◽  
pp. 79-94
Author(s):  
E.K. Macrae ◽  
J.K. Spitznagel
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Cationic Protein ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

Ultrastructural localization of cationic proteins in human polymorphonuclear leukocytes

1978 ◽  
Vol 30 (1) ◽  
pp. 21-35
Author(s):  
W.J. Brown ◽  
E.M. Wood
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Osmium Tetroxide ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Mediators Of Inflammation ◽  
Human Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver ◽  
Neutral Proteases

The present investigation is concerned with the use of the post-formalin ammoniacal silver reaction to detect the arginine-rich cationic proteins in human polymorphonuclear leukocytes at the ultrastructural level. These proteins appear to function as neutral proteases in antibacterial action and as mediators of inflammation. Originally, the ammoniacal silver reaction relied upon primary fixation in dilute formalin which prevented optimum fixation of tissues. This study shows that by using the proper sequence of glutaraldehyde fixation and the ammoniacal silver solution in conjunction with osmium tetroxide treatment, better fixation of the tissue and localization of the ammoniacal silver reaction can be achieved. Also, the ammoniacal silver reaction in human polymorphonuclear leukocytes is exclusively located in the large, crystalline cytoplasmic granules of eosiniphils. All other cytoplasmic granules of neutrophils, eosinophils, and basophils were found to be devoid of the ammoniacal silver reaction product. These results are contrary to previously published experimental data. Possible explanations for this discrepancy are discussed.

scholarly journals Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils and mast cells.

1980 ◽  
Vol 28 (3) ◽  
pp. 238-244 ◽  
Author(s):  
P F Pimenta ◽  
M A Loures ◽  
W de Souza
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Basic Protein ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Cytoplasmic Granules ◽  
Basic Proteins ◽  
Intense Reaction ◽  
The Matrix ◽  
Ammoniacal Silver

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) methods were used to localize basic protein at the ultrastructural level in cytoplasmic granules of rat eosinophils and mast cells isolated using a Metrizamide gradient. Intense reaction was seen in the granules of EPTA-treated eosinophils. Following incubation of the cells for 2 hr in EPTA alone, the matrix was stained. After longer incubation (10 hr), however, both the matrix and core were stained. Cytoplasmic granules of the mast cell show a slight or negative reaction with EPTA. With the AS technique, a large number of silver particles were seen in the nucleus of both eosinophils and mast cells. The mast cell cytoplasmic granules showed intense reaction, while those from eosinophils showed no clear reaction. Acetylation of the cells under conditions sufficient to block most free amino groups prio to EPTA or AS treatment greatly reduced (EPTA) or abolished (AS) the reaction. The results indicate 1) that eosinophil granules contain basic proteins both in the matrix and the core, 2) that the mast cell granules contain a basic protein (probably the alpha-chymotrypsin-like enzyme), which reacts strongly with AS, and 3) that the AS and EPTA methods have different specificities.

scholarly journals POLYMORPHONUCLEAR LEUKOCYTES IN IMMUNOLOGIC REACTIONS

1966 ◽  
Vol 124 (4) ◽  
pp. 733-752 ◽  
Author(s):  
Charles G. Cochrane ◽  
Barbara S. Aikin
Keyword(s):  
Basement Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Electron Microscopic Observation ◽  
Severe Damage ◽  
Electron Microscopic ◽  
Cationic Proteins ◽  
Vascular Basement Membrane ◽  
Arthus Phenomenon ◽  
Immunologic Reactions

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.

Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation

1975 ◽  
Vol 18 (1) ◽  
pp. 123-132
Author(s):  
V.O. Sing ◽  
S. Bartnicki-Garcia
Keyword(s):  
Electron Microscopy ◽  
Cell Adhesion ◽  
Cyst Wall ◽  
Cell Attachment ◽  
Sodium Dodecyl ◽  
Film Surface ◽  
Phytophthora Palmivora ◽  
Thin Sections ◽  
Electron Dense Deposit ◽  
Dense Deposit

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration.

scholarly journals Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes

1978 ◽  
Vol 77 (1) ◽  
pp. 59-71 ◽  
Author(s):  
JM Robinson ◽  
RT Briggs ◽  
MJ Karnovsky
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Polymorphonuclear Leukocytes ◽  
Ultrastructural Localization ◽  
H2o2 Production ◽  
Acid Oxidase ◽  
Resting Cells ◽  
Amino Acid Oxidase ◽  
Human Polymorphonuclear Leukocytes

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.

scholarly journals Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes.

1979 ◽  
Vol 150 (3) ◽  
pp. 703-708 ◽  
Author(s):  
Y Rikihisa ◽  
S Ito
Keyword(s):  
Guinea Pig ◽  
Cell Cultures ◽  
Polymorphonuclear Leukocyte ◽  
Polymorphonuclear Leukocytes ◽  
Intracellular Localization ◽  
The Other ◽  
Cytoplasmic Organelles

Rickettsia tsutsugamushi (Gilliam strain) was serially propagated in BHK-21 cell cultures and incubated with guinea pig peritoneal polymorphonuclear leukocytes to study the ultrastructural features of rickettsial uptake and entry into the leukocytes. Significant numbers of rickettsiae were phagocytized selectively by these leukocytes within 30 min. About one-half of these rickettsiae remained sequestered in phagosomes but the other one-half were free from the phagosome and localized directly in the polymorphonuclear leukocyte cytoplasm. Various stages of rickettsial release from the phagosomes were observed. Once free within the polymorphonuclear leukocyte cytoplasm, the rickettsiae were preferentially localized in the glycogen-packed areas which are devoid of lysosomes and other cytoplasmic organelles. This study indicates that rickettsiae phagocytized by polymorphonuclear leukocytes can escape from the phagosome into the cytoplasm.

Resistance and Susceptibility of Mice to Bacterial Infection: Histopathology of Listeriosis in Resistant and Susceptible Strains

1980 ◽  
Vol 30 (3) ◽  
pp. 851-861
Author(s):  
Thomas E. Mandel ◽  
Christina Cheers
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Single Gene ◽  
Cell Types ◽  
Thymus Atrophy ◽  
Monocytes And Macrophages ◽  
Liver Morphology ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
High Doses ◽  
Neutrophil Response ◽  
Red Pulp

C57BL/10 mice have previously been shown to be 100 times more resistant to intravenously injected Listeria monocytogenes than are BALB/c mice due to the action of a single gene, Lr. Differences in the histopathology of listeriosis in the two strains were sought. Of the tissues examined, only liver, spleen, blood, and thymus showed changes. In the liver, Listeria localized in Kupffer cells within 3 h of infection. By 24 h these cells became surrounded by neutrophilic polymorphonuclear leukocytes. After high doses of Listeria , the susceptible BALB/c mice showed many foci surrounded by few polymorphs, whereas in the resistant C57BL/10 mice there were relatively few foci surrounded by many polymorphs. By 4 days in sublethally infected mice the polymorphs in the liver of both strains were being replaced by monocytes and macrophages. Liver morphology returned to normal by 8 days postinfection. In the blood of both strains there was a rise in total lymphocyte numbers at 24 h, followed by a fall in T-lymphocytes and recovery at 5 days. C57BL/10 mice showed an early monocytic response in the blood, whereas BALB/c mice showed a polymorph leukocytosis. In the spleens of both C57BL/10 and BALB/c mice there was an early neutrophil response and red pulp hyperemia. This was followed by a dramatic lymphocyte depletion in the T-dependent periarteriolar regions in both strains beginning 2 days after infection. Absolute numbers of Thy-1 + cells in spleen cell suspensions also fell to 10% of normal, recovering 6 to 8 days postinfection. Surface immunoglobulin-positive B-lymphocytes and Thy-1 − , immunoglobulin-negative “null” cells rose in both strains at days 4 to 5, returning to normal levels on days 10 to 12. Whether the null cells represent lymphocytes or other cell types remains unresolved. Thymus atrophy was seen in the BALB/c mice but not in C57BL/10 mice.

scholarly journals Cationic Proteins of Human Granulocytes. II. Separation of the Cationic Proteins of the Granules of Leukemic Myeloid Cells

Blood ◽  
1974 ◽  
Vol 44 (2) ◽  
pp. 235-246 ◽  
Author(s):  
I. Olsson ◽  
P. Venge
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Aminocaproic Acid ◽  
Cationic Protein ◽  
Cationic Proteins ◽  
Molecular Weights ◽  
Electrophoretic Mobilities ◽  
Human Granulocytes ◽  
Protein Components

Abstract The highly cationic proteins of human granulocytes, whose electrophoretic mobilities toward the cathode are faster than that for lysozyme, were isolated from the cytoplasmic granules of leukocytes, obtained from patients with chronic myeloid leukemia. The granule extract was subjected to chromatography on Sephadex G-75 and E-aminocaproic acid-Sepharose ion adsorbant followed by preparative electrophoresis on agarose. Seven cationic protein components were identified, and five of these were obtained in a pure form. One group of cationic proteins, including components 1-4, exhibited molecular weights in the range 25,500-28,500, almost identical amino acid composition, and complete immunologic identity. Another group of proteins, including components 5-7, exhibited molecular weights in the range 21,000-29,000 and also showed complete immunologic identity; amino acid analysis performed on component 5 indicated a different amino acid composition from that of components 1-4. Cationic proteins with similar electrophoretic mobilities and immunochemical identities were also detected in granule extracts of granulocytes from healthy individuals. The proteins isolated from human granulocytes have a higher molecular weight and a lower content of basic amino acids than the cationic proteins with antibacterial and permeability-increasing properties previously demonstrated in rabbit polymorphonuclear granulocytes.

scholarly journals Brief Report: Ultrastructural Localization of Myeloperoxidase in Human Neutrophil and Rabbit Heterophil and Eosinophil Leukocytes

Blood ◽  
1968 ◽  
Vol 32 (6) ◽  
pp. 935-944 ◽  
Author(s):  
W. B. DUNN ◽  
J. H. HARDIN ◽  
S. S. SPICER
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Human Neutrophil ◽  
Polymorphonuclear Leukocytes ◽  
Ultrastructural Localization ◽  
Human Monocytes ◽  
Specific Granules ◽  
Human Polymorphonuclear Leukocytes

Abstract Ultrastructural cytochemical observations revealed peroxidase reactivity in primary (azurophil) granules, but not in secondary (specific) granules, of rabbit and human polymorphonuclear leukocytes. Peroxidase reactivity was also observed in the rough endoplasmic reticulum and granules of rabbit eosinophils and in granules of human monocytes.

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