Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells. Application of fluorochrome and extracellular quenching technique.
Simultaneous assessment of the total number of bacteria (TNB) ingested, phagocytosis (Ph), phagocytic index (PI), and intracellular bactericidal activity (ICBA) of human monocytes was done by applying the fluorochrome acridine orange technique. Living bacteria stained orthochromatically green, whereas the dead ones were metachromatically red. The stain of extracellular bacteria was completely quenched by crystal violet counterstain. Using the Hypaque-Ficoll separation method combined with glass adherence, the yield of monocytes was 84 +/- 11%, the purity 90 +/- 8%, and the viability 99 +/- 1%. After 60 min of incubation of monocytes with Staphylococcus aureus, phagocytosis was 94 +/- 4%, PI 10.0 +/- 0.5, ICBA 76 +/- 5%, and TNB ingested 946 +/- 67/100 cells. E. coli B4 was equally ingested by PMNs and monocytes and killed intracellularly more efficiently by the latter type of cells. Over the ratios of bacteria to cells of 5:1 to 20:1, phagocytic activity of monocytes was equal or superior to that of PMNs. Phagocytic and bactericidal activities were enhanced by AB serum, more by the fresh one than by inactivated. Phagocytic activity of monocytes was markedly influenced by temperature of incubation. Room temperature (24 degrees C) significantly suppressed phagocytosis. Contrary to the previous beliefs no significant quantitative differences were found between phagocytic and bactericidal functions of monocytes as compared to polymorphonuclear phagocytes. The acridine orange-crystal violet method is simple, reliable, reproducible, and can be used for assessment of functional capacity of human phagocytes.
Pruzanski
W
W
Immunology Diagnostic & Research Centre, Wellesley Hospital, University of Toronto, Ontario, Canada.
Saito
S
S
D000165:Acridine Orange; D000328:Adult; D001770:Blood Bactericidal Activity; D005260:Female; D005840:Gentian Violet; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology*; D010587:Phagocytosis; D013696:Temperature
D000165:Acridine Orange; D005840:Gentian Violet