scholarly journals Enhancement by double-stranded polyribonucleotides of production by cultured mouse peritoneal macrophages of differentiation-stimulating factor(s) for mouse myeloid leukaemic cells

1978 ◽  
Vol 176 (3) ◽  
pp. 665-669 ◽  
Author(s):  
M Tomida ◽  
K Takenaga ◽  
Y Yamamoto ◽  
M Hozumi
Keyword(s):  
Cell Line ◽  
Culture Medium ◽  
Optimal Dose ◽  
Total Activity ◽  
Peritoneal Macrophages ◽  
Leukaemic Cell ◽  
Cell Lysate ◽  
Leukaemic Cells ◽  
Mouse Peritoneal Macrophages ◽  
Stimulating Factor

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.

Renin and angiotensins in cultured mouse adrenocortical tumour cells

1985 ◽  
Vol 108 (4) ◽  
pp. 545-549 ◽  
Author(s):  
Mitsuhide Naruse ◽  
Kazuo Shizume ◽  
Tadashi Inagami
Keyword(s):  
Adrenal Gland ◽  
Substrate Specificity ◽  
Cell Line ◽  
Cathepsin D ◽  
Culture Medium ◽  
Renin Activity ◽  
Cell Lysate ◽  
Specific Affinity ◽  
Adrenocortical Tumour ◽  
Optimal Ph

Abstract. Mouse adrenal tissue has been reported to contain high renin activity. However, it is not clear whether the renin is produced inside the tissue or is derived from a blood-borne component. We have investigated a cloned cell line of mouse adrenocortical tumour (Y-1) which has a steroidogenic activity. Sizable quantities of renin were demonstrated, predominantly in the cell lysate. This renin activity was distinguished from cathepsin D in view of its specific affinity to anti-renin antibody, optimal pH was determined, and the substrate specificity was checked with haemoglobin. Immunoreactive angiotensins were also detectable, but were demonstrated both in the cell and in the culture medium. This study provides further evidence for the existence of renin intrinsic to the adrenal gland. This study also suggests an intracellular role for renin and possible secretion of generated angiotensins.

scholarly journals Mouse macrophage elastase. Purification and characterization as a metalloproteinase

1981 ◽  
Vol 193 (2) ◽  
pp. 589-605 ◽  
Author(s):  
M J Banda ◽  
Z Werb
Keyword(s):  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
Serine Proteinases ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Predominant Form ◽  
Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

scholarly journals Dynamics of leukotriene C production by macrophages.

1980 ◽  
Vol 152 (5) ◽  
pp. 1236-1247 ◽  
Author(s):  
C A Rouzer ◽  
W A Scott ◽  
A L Hamill ◽  
Z A Cohn
Keyword(s):  
Time Course ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Experimental Conditions ◽  
Silicic Acid Chromatography ◽  
Pg Release ◽  
Antigen Antibody ◽  
Mouse Peritoneal Macrophages ◽  
Hydroxyeicosatetraenoic Acids

A method for the radiochemical assay of LTC production by mouse peritoneal macrophages in vitro is presented. The method involves labeling macrophages in culture with [5,6,8,9,11,12,14,15-3H]20:4 followed by stimulation of arachidonic acid (20:4) release under the experimental conditions desired. Radiolabeled leukotriene C (LTC) is recovered from the culture medium by extraction and silicic acid chromatography in 40% yield with full retention of biological activity. Because this LTC is radiochemically pure, the quantity of LTC release may be estimated from the amount of radioactivity in the sample. Use of the radioassay to study parameters affecting LTC synthesis by macrophages indicated that the time course of LTC synthesis and its relationship to the dose of a phagocytic stimulus (zymosan) were very similar to those of prostaglandin (PG) release. LTC release was also similar to that of PG in that lower levels of both metabolites were produced by Corynebacterium parvum-elicited macrophages than by resident cells. Finally, LTC release was stimulated in response to a challenge with antigen-antibody complexes, but lower maximal levels were attained than those with zymosan. The data presented here are consistent with the hypothesis that challenge of macrophages with a phagocytic stimulus leads to the release of 20:4 by an inducible phospholipase. Cyclooxygenase and lipoxygenase then compete for the released 20:4, leading to the production of PG, hydroxyeicosatetraenoic acids, and LTC.

Dexamethasone and Phorbol Ester, but not Cytokines, Increase the Production of Plasminogen Activator Inhibitor Type-2 in the PL-21 Human Promyelocytic Leukemia Cell Line

1991 ◽  
Vol 66 (02) ◽  
pp. 232-238 ◽  
Author(s):  
Kenji Niiya ◽  
Tetsuo Takeuchi ◽  
Makoto Kobayashi ◽  
Isao Miyoshi ◽  
Tomohiro Hayashi ◽  
...  
Keyword(s):  
Cell Line ◽  
Culture Medium ◽  
Leukemia Cell ◽  
Plasminogen Activator Inhibitor ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Lysate ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
Promyelocytic Leukemia Cell Line

SummaryPL-21 is a promyelocytic leukemia cell line that produces plasminogen activator inhibitor 2 (PAI-2). Differentiation-linked expression of PAI-2 was investigated by adding cell-differentiation promoting agents [such as phorbol myristate acetate (PMA), retinoic acid (RA), dexamethasone (Dex), and recombinant cytokines, including tumor necrosis factor-α (TNF-a), transforming growth factor-β (TGF-β), granulocyte-colony stimulating factor (G-CSF), and interleukin-6 (IL-6)] into the culture medium of PL-21 cells. PAI-1 and PAI-2 antigens were measured by an enzyme-linked immunoassay. The PAI-1 antigen, however, became detectable only after stimulation. The presence of PAI-2 antigen was further verified by immunoblotting using a monoclonal antibody against PAI-2 purified from a PL-21 culture medium. PAI activity both in the culture medium and in the cell lysate increased approximately 70-fold after exposure to PMA. Both PAI-1 and PAI-2 antigens increased, but the amount of the latter in the culture medium and in the cell lysate was approximately 10 times and 2,500 times, as much, respectively, as that of the former. Dex also increased the intracellular PAI activity approximately 6-fold, parallel with PAI-2 antigen. PAI-1 antigen increased only slightly in the culture medium but not in the cell lysate after Dex-stimulation. As with the case of PMA, TNF-a and IL-6 induced PL-21 cells to macrophage-like cells, but did not affect the PAI activity. Thus, the increase of the PAI-2 production by PMA may not necessarily depend on differentiation into macrophages. Other cytokines examined did not increase the PAI activity. Glucocorticoid has various effects on the fibrinolytic system because it has been also shown that the mRNA of PAI-2 in a human fibrosarcoma cell line decreases in response to Dex.

scholarly journals Infectivity of amastigotes of Trypanosoma cruzi

1986 ◽  
Vol 28 (4) ◽  
pp. 205-212 ◽  
Author(s):  
Tecia Ulisses de Carvalho ◽  
Wanderley de Souza
Keyword(s):  
Trypanosoma Cruzi ◽  
Guinea Pig ◽  
Cell Line ◽  
Peritoneal Macrophages ◽  
Mouse Peritoneal Macrophages ◽  
Pig Serum

The infectivity amastigotes of Trypanosoma cruzi, isolated from the supernatant of the J774G8 macrophage-like cell line infected with trypomastigotes to normal macrophages in vitro was tested. After a period of 1 h of T. cruzi-macrophage interaction about 2% of the mouse peritoneal macrophages had ingested amastigotes. In contrast 12% of the macrophages had ingested epimastigotes. Treatment of the amastigotes with trypsin did not interfere with their ingestion by macrophages. Once inside the macrophages the amastigotes divided and after some days transformed into trypomastigotes. When i.p. inoculated into mice the amastigotes were highly infective, inducing high levels of parasitaemia and tissue parasitism. As previously described for trypomastigotes, amastigotes were not lysed when incubated in the presence of fresh guinea-pig serum. Contrasting with what has been described for trypomastigotes, the resistance of amastigotes to complement-mediated lysis persisted after treatment with trypsin.

scholarly journals THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

1972 ◽  
Vol 55 (1) ◽  
pp. 186-204 ◽  
Author(s):  
Ralph M. Steinman ◽  
Zanvil A. Cohn
Keyword(s):  
Horseradish Peroxidase ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Wide Range ◽  
Mouse Macrophages ◽  
Secondary Lysosomes ◽  
Endocytic Vesicles ◽  
In Vitro Interaction ◽  
Mouse Peritoneal Macrophages

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 106 cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-125I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-125I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.

scholarly journals Hydrophobic adsorption chromatography of colony-stimulating activities and erythroid-enhancing activity from the human monocyte-like cell line, GCT

Blood ◽  
1981 ◽  
Vol 58 (6) ◽  
pp. 1148-1154 ◽  
Author(s):  
CN Abboud ◽  
JK Brennan ◽  
GH Barlow ◽  
MA Lichtman
Keyword(s):  
Cell Line ◽  
Culture Medium ◽  
Human Monocyte ◽  
Colony Growth ◽  
Human Cell Line ◽  
Adsorption Chromatography ◽  
Colony Stimulating Factor ◽  
Serum Free ◽  
Hydrophobic Adsorption ◽  
Stimulating Factor

Abstract The human cell line, GCT, secretes hemopoietins into serum-free culture medium. The conditioned medium contains activities that stimulate neutrophil-monocyte, macrophage, eosinophil, and erythroid colony growth in human marrow cultures. We have used hydrophobic adsorption chromatography to separate a neutrophil-monocyte colony-stimulating factor (CSF) from the other colony-stimulating activities. This hydrophobic CSF has no eosinophil-stimulating activity and is virtually devoid of erythroid-stimulating activity.

Action Of Ticlopidine On The Oxygenated Metabolism Of Arachidonic Acid In The Mouse Peritoneal Macrophages

1981 ◽  
Author(s):  
M Rigaud ◽  
H Rabinovitch ◽  
J Durand ◽  
J C Breton ◽  
G Rigaud
Keyword(s):  
Arachidonic Acid ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Eicosatrienoic Acid ◽  
Peritoneal Macrophages ◽  
Membrane Phospholipids ◽  
Glass Capillary ◽  
Drug Concentrations ◽  
High Concentrations ◽  
Mouse Peritoneal Macrophages

When ticlopidine is added to macrophages cultures, in the absence of exogenous arachidonic acid, there is a production of both“prostanoids” and“eicosanoids” in the culture medium. These products have been measured using glass capillary gas chomatography prior to multiple ion mass spectrometry. The quantitative determinations are made 5, 15, 25, 35 and 45 minutes after the drug was added to the macrophages cultures. The three drug concentrations used (10-4M, 5.10-5M and 2.5 10-5M) induce a liberation of 6-keto-PGF1α in the culture medium. As in our system 6-keto-PGF1α seems to be the major metabolite of PGI2, ticlopidine is likely to act by releasing important quantities of PGI2 in macrophages. These results suggest an increase of liberation of the endogenous arachidonic acid from the membrane phospholipids of the macrophages or a lack in the acyltransferase system of the cell membranes. The lipoxygenasic pathway was also studied. When ticlopidine is added to macrophages, two products are liberated: . 12-HETE as measured by single iondetection . and 10-hydroxy-ll-12-epoxy, -5, 8, 14-eicosatrienoic acid which comes from an internal rearrangement of the 12-HPETE. In these results, there is a discrepancy between the fact that ticlopidine increases the concentration of 12-HETE and surely its precursor the 12-HPETE and the fact that the synthesis of PGI2 is not inhibited by these high concentrations of hydroperoxide. To understand this phenomenon we studied the production of hydroperoxide when arachidonic acid is incubated with soybean lipoxygenase. When ticlopidine (10-4M) is added to the reaction mixture (AA + soy lipoxygenase) there is an increase of the initial rate and an extent of the reaction as if the enzyme irreversible deactivation was postponed. Ticlopidine could act by suppressing the classical inhibition of PGI2 synthetase by hydroperoxides, in particular 12-HPETE and 15-HPETE, both produced by mammalian cells.

scholarly journals Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

1982 ◽  
Vol 206 (2) ◽  
pp. 231-237 ◽  
Author(s):  
Ulf Lindahl ◽  
Svein O. Kolset ◽  
Jarl Bøgwald ◽  
Bjarne Østerud ◽  
Rolf Seljelid
Keyword(s):  
Culture Medium ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Peritoneal Macrophages ◽  
Enzyme Concentration ◽  
Factor X ◽  
Peptide Substrate ◽  
Substrate Peptide ◽  
Vipera Russelli ◽  
Mouse Peritoneal Macrophages

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 106cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.

scholarly journals The ingestion and digestion of human lactoferrin by mouse peritoneal macrophages and the transfer of its iron into ferritin.

1977 ◽  
Vol 146 (3) ◽  
pp. 817-827 ◽  
Author(s):  
J L van Snick ◽  
B Markowetz ◽  
P L Masson
Keyword(s):  
Iron Content ◽  
Culture Medium ◽  
Reticuloendothelial System ◽  
Peritoneal Macrophages ◽  
Human Lactoferrin ◽  
Saturation Point ◽  
Intracellular Digestion ◽  
Additional Support ◽  
Mouse Peritoneal Macrophages

Human lactoferrin (Lf) labeled with 125I and/or 59Fe was found to be ingested in vitro by mouse peritoneal macrophages (MPM). The uptake measured after 15 h incubation reached a saturation point at a concentration of 200 microgram/ml in the culture medium, whatever was the iron content of Lf. In such conditions, the uptake of transferrin (Tf) used as a control was 10 times lower. At a concentration of 80 microgram/ml in the medium, one cell picked up about 0.7 X 10(6) molecules of Lf per hour, and 0.13 X 10(6) molecules of Tf per hour. Iron-saturated Lf disappeared from MPM with a half life of 14.5 h, whereas the halflife of iron-free Lf was 4.2 h. Concomitant with the intracellular digestion of Lf, the iron was transmitted to ferritin. These data provide additional support for the hypothesis that Lf plays a key role in iron turnover, especially at the level of the reticuloendothelial system where iron is recovered from the catabolism of erythrocytes.

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