Monocyte Suspensions Research Articles

Identification of monocytes in suspensions of mononuclear cells

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.

Studies on human monocytes with a multiparameter cell sorter.

Suspensions of human lymphocytes and monocytes separated by the Ficoll-hypaque method from the peripheral blood show a Coulter volume distribution, measured with a multiparameter cell sorter, characterized by a minor peak at 500 mu3, containing 5-15% of the cells, and a major peak at 200 mu3. Using fluorescent latex particles we have found that the monocytes, the cells that ingest the latex particles, all lie in the 500 mu3 peak; conversely, all of the cells in the 500 mu3 peak are monocytes. When the cell suspensions are incubated, the monocytes increase both the average volume and in absolute numbers. The number of monocytes approximately doubles during 3 days of incubation, when it reaches its maximum value. At that time we have found that all of the monocytes lack receptors for sheep red blood cells and all possess receptors for human gamma-globulin. The increase in monocyte number appears, therefore, to arise from the enlargement of "monocyte presursors" that resemble lymphocytes in volume and resemble both the monocytes and the B lyphocytes with respect to surface sheep red blood cell and human gamma-globulin receptors.

Antibody-dependent human lymphocytotoxicity: a micro assay system.

A micro assay system is described for measuring antibody-dependent lymphocytotoxicity with human effector cells. The target cells were mouse mastocytoma cells sensitized with rabbit antibody. The tests were performed in microtiter plates, each well containing 5 x 10(3) 51Cr-labeled target cells, together with varying numbers of effector cells. Whereas highly purified lymphocyte suspensions induced lysis, purified monocyte and granulocyte suspensions had virtually no effect. In parallel experiments it was shown that the latter cell suspensions were very active in lysing sensitized human erythrocytes, which in turn were not lysed by purified lymphocytes. Finally, it was shown that the lytic capacity of lymphocytes was not affected by preservation of the cells at liquid nitrogen temperature. It is concluded that the system described can be used for the clinical analysis of antibody-dependent lymphocytotoxicity in lymphocyte suspensions.

The endotoxin-induced coagulant activity of human monocytes.

Leucocyte suspensions, exposed to endotoxin in vitro, develop coagulant activity which has been identified as tissue factor. Pure suspensions of polymorphonuclear (PMN) neutrophils, lymphocytes and monocytes were exposed to endotoxin and tested for tissue factor activity after 4 h incubation at 37 degrees C. The results indicate that the monocyte is the cell primarily responsible for the endotoxin-induced coagulant activity of mixed leucocyte suspensions, the small amount of activity demonstrated in PMN neutrophil and in lymphocyte suspensions at high cell concentrations being accounted for by monocyte contamination of less than 1%.

Monocyte Function in Children With Neutropenia and Chronic Infections

Abstract The phagocytic, bactericidal, and metabolic activities of monocytes from patients with congenital neutropenia were not significantly different than the activities of monocytes from patients with cyclic neutropenia or chronic infections. However, significant differences in metabolic, phagocytic, and bactericidal functions were found between monocytes from any source and normal neutrophils. During phagocytosis, monocytes produced more of the bactericidal product hydrogen peroxide than did neutrophils. However, monocytes in suspension had decreased phagocytic capacity compared to neutrophils and killed staphylococci less well. Moreover, monocytes had a diminished capacity to iodinate ingested bacteria. Quantitation of granular myeloperoxidase activity revealed that monocytes had a relative deficiency of this enzyme, which could explain the decreased iodination and killing of ingested bacteria by monocytes when compared to neutrophils. These findings offer an explanation for the failure of compensatory monocytosis to protect the host from bacterial infection when neutrophil reserves are diminished.

Studies on tubercle bacillus-monocyte relationship. I. Quantitative analysis of effect of serum of animals vaccinated with BCG upon bacterium-monocyte system.

Studies of the conditions necessary for maintenance of constant cell populations in vitro in the Mackaness type of culture chamber have indicated the importance of preliminary trypsinization of cells and the beneficial effect of 40 per cent rabbit serum in Tyrode's solution. Under these optimal conditions, uninfected suspensions of monocytes exhibited little change in cell numbers over a period of 40 to 72 hours. Infection of monocytes with the virulent H37Rv strain of tubercle bacillus resulted in an early degeneration of a certain proportion of the cells cultivated in the presence of normal rabbit serum. This degeneration was apparent not only for cells of tuberculin-negative animals but also for those derived from tuberculin-positive animals. The serum of animals vaccinated with BCG exerted a favorable effect upon the survival of monocytes infected with virulent tubercle bacilli. Treatment with this serum caused a delay in degeneration of infected normal cells (cells of tuberculin-negative rabbits) and a complete inhibition of degeneration of infected immune cells (cells of rabbits vaccinated with BCG).