Bactericidal Activity of Specific and Azurophil Granules from Human Neutrophils: Studies with Outer-Membrane Mutants of Salmonella typhimurium LT-2

Abstract

Extracts of specific granules and azurophil granules from human neutrophils were tested for their bactericidal activity against various lipopolysaccharide mutants of Salmonella typhimurium LT-2. Three purified granule populations, one specific and two azurophil, were obtained by isopycnic centrifugation of homogenized neutrophils. Each was extracted with 0.2 M acetate buffer (pH 4), and the extracts were dialyzed against phosphate-buffered saline (pH 7) to remove acetate. These extracts contained ≥84% of the lysozyme, lactoferrin, or myeloperoxidase initially present in the whole granules. The S. typhimurium mutants possessed Ra, Rc, Rd 1 , Rd 2 , or Re lipopolysaccharide. As the carbohydrate content of the lipopolysaccharide decreased, the bacteria became increasingly more susceptible to the bactericidal activity of all granule extracts. Bactericidal activity of the extracts was in the order: mixed (azurophil + specific) ≥ azurophil » specific. Specific granules were bacteriostatic for S through Rd 2 bacteria. They were bactericidal only for the Re mutant. Both azurophil granule populations were equally bactericidal. Extracts boiled for 30 min retained none of their bactericidal activity for any of the bacteria; however, they remained bacteriostatic for the deep rough (Rd 2 , Re) mutants. Bactericidal activity was dependent upon pH, in that mixed and azurophil granule contents killed the smooth parent and Ra mutant best at pH 5, the Rc and Rd 1 mutants to the same degree at pH 5 to 8, and the deep rough mutants (Rd 2 and Re) best at pH 8. Specific granule contents were most bacteriostatic for S through Rd 2 bacteria at pH 5 and killed the Re mutant only at pH 8. Thus, as the S. typhimurium lipopolysaccharide content decreased, the bactericidal pH optimum increased. Killing by all extracts was dependent upon incubation temperature, with almost no bactericidal or bacteriostatic activity observed when bacteria and granule fractions were incubated on ice (2°C) and plated immediately. Intermediate killing was observed at 22°C. If bacteria were incubated with granule extracts at 2°C, washed free of extract, suspended in medium without extract, and reincubated at 37°C, killing was observed. This suggested that a component(s) of the extracts was sticking to the bacteria at 2°C but killing only at 37°C.