Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Abstract

Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations

“…In line with a complementary role for the NADPH oxidase in this context, S aureus killing was more or less abolished in cells treated with the NADPH oxidase inhibitor diphenylene iodonium, whereas the killing of E coli was only partially impaired (supplemental Figure 8). Similar abnormalities in killing have been observed with neutrophils from patients with chronic granulomatous disease 15 (data not shown). It should be noted that the production of reactive oxygen species by the NADPH oxidase in response to particulate stimuli, such as unopsonized or opsonized zymosan (supplemental Figure 9A), or soluble stimuli, such as PAF/ fMLP (not shown), was not impaired in FHL-5 neutrophils.…”

Killing by neutrophil extracellular traps: fact or folklore?

“…After agitation at medium/high speed for 2 minutes in a microplate mixer, aliquots were taken, sequentially diluted in H 2 O, adjusted to pH 11 and 0.9% NaCl, and plated to determine CFU as described. 5 The results of these experiments are shown in Figure 1A. At the end of the 20-minute incubation with bacteria, DNase I (100 U/mL) was added to the wells not already containing it, and incubation continued for an additional 10 minutes.…”

The role of chloride anion and CFTR in killing ofPseudomonas aeruginosaby normal and CF neutrophils

“…After overnight culture, viable PsA colonies were counted. Preliminary study proved that lysis of the cells in the saponin water solution and dispersion of bacteria by passing through a fine needle was as effective as the recently published alkaline water method [34].…”