Quantitative Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis

Abstract

A B S T R A C T Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red 0 emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by Nethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed. INTRODUCTIONPhagocytosis is an integrated series of complex events.Certain as yet unidentified characteristics of the surface of a particle cause it to be bound to the plasma membrane of a polymorphonuclear leukocyte which then internalizes it. As the phagocytic vesicle is formed and moves centripetally, lysosomal granules discharge their contents into it and disappear from the cytoplasm, a process termed degranulation. These processes are accompanied by several alterations in the metabolism of the phagocytic cell which have been termed metabolic concomitants of phagocytosis. One of these is an increase in oxidatio...

Effects of 2-{4-(2-Imidazo[1,2-a]Pyridyl)Phenyl} Propionic Acid (Y-9213) and Anti-Inflammatory Drugs on Erythrocytes, Polymorphonuclear Leukocytes and Lysosomes in Vitro

“…Similarly, colchicine and theophylline were reported to inhibit the secretion of lysosomal enzymes (12), and phagocytosis (11). In the present experiment (Table 10), colchicine, isoproterenol, theophylline and papaverine inhibited phagocytosis of the particles.…”

Reversible phagocytosis in rabbit polymorphonuclear leukocytes.

“…It must be emphasized however that the processing of ingested particles, indicate(d by lysosomal frision with the phagocytic vesicle, occurs rapidly (4). DISCUSSION The foregoing results establish the exocytosis of previously phagocytized particles by rabbit PMN.…”

“…The use ofthe Coulter counter for assay ofphagocytosis with whole yeast cell has been recently reported (5), but the volume changes obtained are complicated in the presence of particles that may be absorbed to the surface. Absorption ofoil emulsion as indicated (4) Unlike phagocytosis itself, reversal does not require exogenous divalent cations. After 20 min incubation postphagocytosis in medium containing 4 mM EDTA, the fraction of cell-associated oil remaining was 0.59±0.076 (2 SE, eight experiments).…”

Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors.

“…1 and 2). The very early increases in the instantaneous rate of particle uptake and the cumulative rate of particle uptake which plateaus after 5-6 min of phagocytosis correlate temporally with increases in the rate of neutrophil glucose metabolism (25). By analogy, the slower and more sustained stimulation of glucose metabolic rates of neutrophils produced by chemotactic factors (Figs.…”