Subcellular location and properties of bactericidal factors from human neutrophils

We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins.