Effects of Pseudomonas aeruginosa cytotoxin on human serum and granulocytes and their microbicidal, phagocytic, and chemotactic functions

Abstract

The effect of Pseudomonas aeruginosa cytotoxin on human granulocytes (PMNs) and pooled human serum was studied by hemacytometer counts, phagocytic, bactericidal, and chemotaxis assays, and by transmission electron microscopy. The optimal assay conditions for phagocytosis of 75Se-labeled P. aeruginosa 1348A included 20% pooled human serum and a ratio of one PMN to between 10 and 20 bacteria. For the bactericidal assay, 20% pooled human serum and a ratio of one PMN to between one and five bacteria were used. Chemotaxis of PMNs was studied by agarose gel technique with 10(-7) M f-Met-Leu-Phe or 0.01 to 35 micrograms of cytotoxin per ml as a chemoattractant. The degree of PMN destruction was dependent on cytotoxin concentrations and PMN exposure time to cytotoxin. Virtually complete PMN lysis was observed after a 2-h exposure to 6 to 10 micrograms of cytotoxin per ml. PMN exposure to 2 micrograms of cytotoxin per ml for as long as 2 h had no adverse effect on phagocytosis. PMN exposure to greater than or equal to 4 micrograms of cytotoxin per ml for 2 h demonstrated a significant decrease in the percentage of bacteria killed. The results of experiments designed to separate cytotoxin effect on PMN lysis from the effect on PMN bactericidal capacity showed that there is an effect of cytotoxin on PMN bactericidal function. PMN exposure to 4 micrograms of cytotoxin per ml for 30 min caused a significant decrease in PMN migration. Cytotoxin had no chemoattractant qualities or effect on pooled human serum as studied by chemotaxis and phagocytosis assays. Although a cytotoxin concentration of greater than or equal to 2 micrograms/ml was required to demonstrate PMN ultrastructural changes observed in transmission electron microscopy studies, at a concentration of 0.1 microgram/ml, cytotoxin caused an impairment in the integrity of the PMN membrane, allowing a low-molecular-weight substance (ruthenium red) to enter into the cytoplasm. Cytotoxin may be an important factor in the pathogenesis and in the high mortality rate of patients with P. aeruginosa infections.