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1.
Med Hypotheses ; 53(2): 107-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10532700

ABSTRACT

Cytokines and their effects are widely studied in immunology, but the cellular mechanisms responsible for producing these substances are not yet described or proven. Antigen presenting cells (APCs), macrophages and dendritic cells are suitable subjects for such a study. One of the many papers illustrating the interplay between phagocytosis, cytokine secretion, and cell adhesion is one by Kitajima et al. The intracellular processes involve both cytokine secretion and mobilization of the membrane system of the cell. Five pieces of evidence are presented to propose that the Golgi complex is the source both of the membrane translocated and cytokine production. Morphologic evidence is cited that reverse pinicytosis does not play a significant role in the mouse macrophage system.


Subject(s)
Cytokines/metabolism , Immunity, Cellular/physiology , Animals , Humans , Mice
3.
Lab Invest ; 36(3): 310-20, 1977 Mar.
Article in English | MEDLINE | ID: mdl-839741

ABSTRACT

The functional capacity of guinea pig megakaryocytes was tested by studying their ability to concentrate serotonin and their response to agents which trigger the platelet release reaction. Megakaryocytes can concentrate 3H-serotonin as demonstrated by autoradiography after exposure to 0.5 muM 3H-serotonin and by quantitative measurement of isotope incorporation within 60 minutes. Uptake of isotope is rapid and linear within the first 30 minutes and tapers off between 30 and 60 minutes. Incorporation of isotope is diminished during exposure to cold, 2 muM reserpine, and 20 muM imiprimine. The following triggering agents: 10(-5) to 10(-3) M ADP, 1 to 100 units of thrombin, 10(-5) to 10(-3)M epinephrine, and 1 to 12 muM ionophore A23187 all produce significant release of stored 3H-serotonin. In the presence of ADP, albumin and serum completely inhibit the release of serotonin. Scanning microscopic studies show that coincident with serotonin release the triggering agents produce marked changes in cell shape. Transmission electron microscopy on these cells shows that there is the appearance of a prominent contraction zone, which is composed of microfilaments, and also variable diminution of cytoplasmic granules. The specifically induced serotonin release from megakaryocytes coupled with shape change and evidence of cell contraction produced by certain agents demonstrate one aspect of the functional similarly between megakaryocytes and platelets.


Subject(s)
Blood Platelets/metabolism , Megakaryocytes/physiology , Serotonin/metabolism , Adenosine Diphosphate/pharmacology , Animals , Autoradiography , Blood Platelets/physiology , Cold Temperature , Cytoplasmic Granules/ultrastructure , Dimethyl Sulfoxide/pharmacology , Epinephrine/pharmacology , Guinea Pigs , Imipramine/pharmacology , Ionophores/pharmacology , Megakaryocytes/drug effects , Megakaryocytes/metabolism , Megakaryocytes/ultrastructure , Microscopy, Electron , Microscopy, Electron, Scanning , Reserpine/pharmacology , Thrombin/pharmacology , Tritium
4.
Lab Invest ; 36(3): 321-8, 1977 Mar.
Article in English | MEDLINE | ID: mdl-320386

ABSTRACT

The capacity of megakaryocytes to take up particles and macromolecules was tested by exposing them to homologous erythrocytes, latex particles, Micrococcus lysodeikticus, and horseradish peroxidase. Uptake of particles 1.1 to 7 mum. in size was extensive under the experimental conditions employed. After uptake of each of the three particles the addition of tannic acid and calcium to the fixative produced electron-dense deposits within the vacuole surrounding the particle; therefore, the vacuole was not closed off. This finding indicates that many of the particles could be actually trapped within the preformed elements of the demarcating membrane system. Horseradish peroxidase was concentrated within cytoplasmic vacuoles after 2 hours of exposure to 0.1 mg. per ml. The presence of surface membrane receptors was determined by the sheep cell rosette method. The presence of the C3b receptor for complement was determined by the sheep cell rosette method. The presence of the C3b receptor for complement produced rosette formation. The functional capacity of megakaryocytes to react like platelets was also tested by observing cell lytic changes and determining that there was approximately 40% release of 3H-serotonin produced by specific rabbit antiguinea pig platelet antiserum in the presence of complement. These studies demonstrate several functional capacities of megakaryocytes: the uptake of particles, pinocytosis, and the cell lytic response to specific antibody.


Subject(s)
Blood Platelets/immunology , Immune Sera/pharmacology , Megakaryocytes/physiology , Phagocytosis , Pinocytosis , Acid Phosphatase/metabolism , Animals , Blood Platelets/metabolism , Complement System Proteins , Erythrocytes , Guinea Pigs , Horseradish Peroxidase , Immune Sera/isolation & purification , Immunologic Techniques , Latex , Megakaryocytes/enzymology , Megakaryocytes/ultrastructure , Micrococcus , Microscopy, Electron , Microspheres , Serotonin/metabolism
5.
J Histochem Cytochem ; 24(4): 601-5, 1976 Apr.
Article in English | MEDLINE | ID: mdl-58024

ABSTRACT

Plasma membranes in isolated guinea pig megakaryocytes and washed platelets are poorly stained with the usual methods used to outline cell membranes. The addition of tannic acid and calcium to the initial fixative is useful to enhance electron density of all surface-derived membrane systems in these cells. The method described here shows that the increased electron denisty of membrane after fixation in the presence of tannic acid occurs both at the cell surface and along the invaginated membrane systems.


Subject(s)
Blood Platelets/ultrastructure , Cell Membrane/ultrastructure , Megakaryocytes/ultrastructure , Animals , Binding Sites , Guinea Pigs , Hydrolyzable Tannins , Methods , Microscopy, Electron , Staining and Labeling
6.
J Cell Biol ; 69(1): 159-72, 1976 Apr.
Article in English | MEDLINE | ID: mdl-3509

ABSTRACT

Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.


Subject(s)
Megakaryocytes , Adenosine , Animals , Bucladesine , Centrifugation , Centrifugation, Density Gradient , Guinea Pigs , Hydrogen-Ion Concentration , Maleimides , Megakaryocytes/ultrastructure , Ouabain , Theophylline
7.
Lab Invest ; 34(4): 440-50, 1976 Apr.
Article in English | MEDLINE | ID: mdl-1263446

ABSTRACT

Recently a new method was developed to culture bone marrow cells in a liquid medium on a glass surface. Two kinds of colonies develop in these cultures, namely mononuclear phagocyte and granulocyte colonies. The study of the ultrastructure of the cells in the mononuclear phagocyte colonies was the primary aim of the present study. The architecture of the mononuclear phagocyte colonies appeared to be quite different from that of the granulocyte colonies, since in the latter, the cells lie close together in dense clusters, whereas in mononuclear phagocyte colonies the cells are more loosely dispersed with the highest cell density at the center and stellate orientation of the cells at the periphery. However, both kind of colonies grow entirely separate from each other and mixed colonies are not observed. Electron microscopy showed that there are three types of cell in the mononuclear phagocyte colonies, i.e., monoblasts, promonocytes, and macrophages. The ultrastructure of the promonocytes and macrophages of the colonies is similar to that of the same types of cell isolated directly from the mouse. The monoblast, the most immature cell of mononuclear phagocyte colonies has not been characterized before. The ultrastructure of this cell is clearly distinct from that of the promonocyte in having a round contour without pseudopods, a nuclear to cytoplasmic ratio greater than one, a cytoplasm that contains many polyribosomes, a few small granules, and a small Golgi complex surrounded by a few short strips of rough endoplasmic reticulum.


Subject(s)
Bone Marrow Cells , Bone Marrow/ultrastructure , Cells, Cultured , Monocytes/ultrastructure , Cell Division , Cell Nucleus/ultrastructure , Cytoplasm/ultrastructure , Granulocytes/ultrastructure , Hematopoietic Stem Cells/ultrastructure , In Vitro Techniques , Macrophages/ultrastructure , Microscopy, Electron, Scanning , Monocytes/enzymology , Peroxidases/metabolism
9.
Blood ; 45(3): 435-49, 1975 Mar.
Article in English | MEDLINE | ID: mdl-46760

ABSTRACT

The iron storage macrophage has been isolated from the marrow of Imferon-treated mice and studied in vitro by morphologic, histochemical, and functional tests and isotope labeling techniques. These macrophages on stained preparations are large, many times binucleate cells (up to 150 mu), and show Prussian blue reactivity. In Epon-embedded, stained thick sections they contain elongated narrow basophilic inclusions. These macrophages are actively phagocytic and pinocytic; histochemical studies show that these cells are rich in acid phosphatase, nonspecific esterase, and PAS diastase-resistant activity. Iron storage macrophages do not incorporate the 3H-thymidine. The electron microscopic appearance of this macrophage shows that the cell has ferritin free in the cytoplasm and several types of cytoplasmic granules: those with large quantities of electron-dense ferritin and/or hemosiderin (type A), elongated granules (type B) with moderately electron dense homogeneous matrix and some ferritin at the periphery, and granules with heterogeneous content (type C). The above findings demonstrate that the iron storage cell is a mature macrophage which contains hydrolases, ferritin, and a unique population of cytoplasmic granules which are lysosomal in nature. There is some evidence to suggest that the unusual lysosome (type B granule) occurs after macrophages have ingested erythrocytes.


Subject(s)
Bone Marrow Cells , Bone Marrow/drug effects , Iron-Dextran Complex/pharmacology , Macrophages/drug effects , Acid Phosphatase/blood , Animals , Autoradiography , Erythrocytes/drug effects , Esterases/blood , Femur , Immune Adherence Reaction , Iron/metabolism , Macrophages/immunology , Macrophages/ultrastructure , Mice , Microbial Collagenase , Microscopy, Electron , Microscopy, Phase-Contrast , Phagocytosis , Specimen Handling , Staining and Labeling , Streptococcus/immunology , Thymidine/metabolism , Tritium , Zymosan
10.
J Cell Biol ; 62(3): 802-14, 1974 Sep.
Article in English | MEDLINE | ID: mdl-4859401

ABSTRACT

Mouse peritoneal macrophages in culture for 24 h were exposed to horse [(55)Fe]ferritin and rabbit antihorse [(55)Fe]ferritin antibody complex and the amount of (55)Fe in the medium was assayed up to 2 days after the pulse uptake. Cell survival was assayed by photographing the same areas of the tissue culture Petri dish on successive days and by counting cell numbers per unit area. In experiments in which quantitative assay for cell death is negligible, about 10-20% of the iron ingested by pinocytosis or phagocytosis is released to iron-free medium containing either freshly dialyzed or deironized newborn calf serum (10%). Over the 2-day postpulse period, iron loss is linear. This loss of iron to the medium is significantly reduced by adding iron-saturated newborn calf serum in the postpulse recovery period. A significant portion of the iron released to the medium is bound to transferrin. When human serum is used in the tissue culture system, similar quantities (10-25%) of the ingested iron are lost to the medium 2 days after the pulse.


Subject(s)
Antigen-Antibody Complex , Ferritins/metabolism , Iron/metabolism , Macrophages/metabolism , Animals , Ascitic Fluid/cytology , Autoradiography , Cell Survival , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Horses/immunology , Iron Radioisotopes , Mice , Phagocytosis , Pinocytosis , Protein Binding , Rabbits/immunology
11.
J Cell Biol ; 57(2): 289-305, 1973 May.
Article in English | MEDLINE | ID: mdl-4348785

ABSTRACT

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.


Subject(s)
Ferritins/isolation & purification , Macrophages/analysis , Animals , Cell Nucleus , Cells, Cultured , Cytoplasm/analysis , Cytoplasmic Granules/analysis , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum , Erythrocytes , Ferritins/biosynthesis , Golgi Apparatus , Histocytochemistry , Inclusion Bodies , Iron Isotopes , Macrophages/metabolism , Mice , Microscopy, Electron , Mitochondria , Peritoneum , Phagocytosis , Pinocytosis , Rabbits/immunology , Rats/immunology , Sheep/immunology , Time Factors
14.
J Exp Med ; 132(4): 794-812, 1970 Oct 01.
Article in English | MEDLINE | ID: mdl-5508378

ABSTRACT

Mouse promonocytes have been identified and studied in cultures of bone marrow cells. These cells have a diameter of 14-20 micro, and in stained preparations reveal a large, indented or folded nucleus, and basophilic, finely granular cytoplasm. The living promonocyte viewed by phase contrast shows additional features: nucleoli, small dense bodies, and vesicles in the cytoplasm adjacent to the nuclear hilus, and slight membrane ruffling. Prominent ultrastructural components of promonocytes include a well developed Golgi apparatus, small numbers of centrosomal granules and vacuoles, extensive ribosomal aggregates, and finger-like projections of the cell surface. Promonocytes engage in pinocytosis and phagocytosis, but they are less active in these functions than are peripheral blood monocytes of peritoneal macrophages. Promonocytes are positive for peroxidase, the reaction product being localized to granules most of which are centrally situated in the cell. Monocytes in blood or in inflammatory peritoneal exudates display much smaller numbers of peroxidase-positive granules, and various types of mature mouse macrophages are peroxidase negative.


Subject(s)
Bone Marrow Cells , Bone Marrow/enzymology , Macrophages/cytology , Macrophages/enzymology , Monocytes/cytology , Monocytes/enzymology , Peroxidases/analysis , Animals , Culture Techniques , Femur , Histocytochemistry , Mice , Microscopy, Electron , Peritoneum/cytology , Phagocytosis , Pinocytosis
17.
J Cell Biol ; 38(3): 615-27, 1968 Sep.
Article in English | MEDLINE | ID: mdl-4874495

ABSTRACT

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.


Subject(s)
Aldehydes , Histological Techniques , Leukocytes/cytology , Osmium , Uranium , Acetates , Culture Techniques , Humans , Methods , Microscopy, Electron
19.
J Cell Biol ; 38(2): 377-91, 1968 Aug.
Article in English | MEDLINE | ID: mdl-4874491

ABSTRACT

Mouse macrophages exposed to 30 microg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1-4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.


Subject(s)
Chloroquine/pharmacology , Cytoplasm/drug effects , Macrophages/drug effects , Acid Phosphatase/analysis , Animals , Cell Membrane , Culture Techniques , Histocytochemistry , Male , Mice , Microscopy, Electron , Microscopy, Phase-Contrast
20.
J Cell Biol ; 38(2): 392-402, 1968 Aug.
Article in English | MEDLINE | ID: mdl-4874492

ABSTRACT

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1-2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.


Subject(s)
Chloroquine/pharmacology , Cytoplasm/drug effects , Macrophages/drug effects , Animals , Culture Techniques , Golgi Apparatus , L Cells , Mice , Microscopy, Electron , Microscopy, Fluorescence , Microscopy, Phase-Contrast , Pinocytosis
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