Kinetics of intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes

Abstract

The intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes was investigated independently of the ingestion of these bacteria. Granulocytes were allowed to phagocytose preopsonized bacteria for only 3 min, after which the noningested bacteria were removed by differential centrifugation and 2 washes. With this technique, the number of viable cell-associated bacteria at the start of the assay, determined after lysis of the granulocytes, includes about 80% intracellular bacteria. Intracellular killing depends on (a) the temperature (no killing occurring at 4 "C, maximal killing at 33-39"C, and a decrease in the capacity of the granulocytes to kill ingested bacteria at temperatures above 42"C), and (b) the number of bacteria ingested (after phagocytosis at bacteria-to-cell ratios of 100 : 1 and 1000 : 1, not all of the ingested bacteria are killed, whereas after phagocytosis at lower bacteria-to-cell ratios, almost all ingested bacteria are killed). To determine the maximum number of bacteria that can be killed by granulocytes, intracellular killing was measured after phagocytosis of bacteria at various bacteria-to-granulocyte ratios in the presence of phenylbutazone, a drug which inhibits killing during the ingestion period. Phenylbutazone proved to be a useful tool in the study of intracellular killing, since this drug provides a reversible inhibition of the killing when granulocytes are incubated in its presence for up to 3 min, whereas after longer incubation, the inhibitory effect is irreversible. Calculation based on the data obtained in this study gave maximum rates of intracellular killing amounting to 3.7 x lo5 bacteriai5 x lo6 granulocytesimin for Staph. aureus and 8.5 x lo5 bacteria/5 x 10' granulocytesimin for E. coli.Using the rate of intracellular killing after phagocytosis at a bacteria-to-granulocyte ratio of 1 : 1 and the rate of ingestion obtained in an earlier study, we were able to compute the theoretical numbers of viable extracellular not (yet) ingested, viable intracellular, killed intracellular and total intracellular bacteria. The theoretical curves fit well with the experimental data.

Evaluation of Intracellular Killing of Bacteria by Enriched Populations of Mouse Peritoneal Exudate Neutrophils

“…The continued presence of a heat-labile substance in normal mouse serum was necessary for optimal kill of intracellular P. mirabilis (Table 3), in keeping with the results of Leijh et al (1981) with E. coli and hum"an neutrophils. The mechanism of this stimulation involves cell membrane receptors (Leijh et al, 1981), and is currently being investigated.…”

“…The continued presence of a heat-labile substance in normal mouse serum was necessary for optimal kill of intracellular P. mirabilis (Table 3), in keeping with the results of Leijh et al (1981) with E. coli and hum"an neutrophils. The mechanism of this stimulation involves cell membrane receptors (Leijh et al, 1981), and is currently being investigated. The methodology described may also be used to measure the influence of soluble products produced by activated cells (lymphokines, monokines), of therapeutic drugs and antibiotics, and of various bacterial products hypothesised to influence neutrophil bactericidal rather than phagocytic ability.…”

“…Previously reported methods for removing extracellular bacteria have included the use of differential centrifugation after a short ingestion period (Leijh et al, 1981). However, with mouse cells we found that differential centrifugation alone was less efficient than Percoll density gradient centrifugation and washing in removing extracellular organisms.…”

Inhibitory Effect of Haptoglobin on Granulocyte Chemotaxis, Phagocytosis and Bactericidal Activity

“…After discarding the supernatant, the pellet was washed twice in ice-cold KB-1 in the same manner. The cells were resuspended in their original volume in KB-1, with or without Hp (0.5 mg/ml), and further incubated at 37 8C with continuous, gentle rotation [22]. Aliquots of 100 ml were harvested after 0, 30, 60 and 90 min.…”

Protease inhibitors decrease IgG shedding from Staphylococcus aureus, increasing complement activation and phagocytosis efficiency

“…One of the important functions of this antibody is opsonizing bacteria and improving their recognition by phagocytic leukocytes (Leijh et al, 1981). IgG can also activate the classical complement pathway leading to additional opsonization by the complement C3b/iC3b fragments and the generation of anaphylatoxins (Joiner et al, 1983).…”