NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes (1)
Neutrophil extracellular traps (NETs) provide a potential mechanism for release of neutrophil-derived mediators in CF (DNA, MPO, HNE).
ELISA methods use anti-MPO or anti-HNE capture antibodies and a horseradish peroxidase- conjugated anti-DNA detection antibody. A limited DNAse diges- tion step to cut the long neutrophil DNA strings into pieces small enough to be harvested but large enough to allow antibody binding.
Unstimulated neutrophils released only low amounts of DNA whereas PMA treatment resulted in robust DNA release. Increasing concentrations of DNAse not only resulted in decreased average DNA size but also increased the DNA yield harvested. The 1 ug/ml DNAse dose to be optimal. When digested with this DNAse concentration, DNA is chopped up into fragments of a few kb size resulting in the high- est ELISA signal. Higher DNAseI (30 and 100 ug/ml) doses completely digest the DNA, preventing antibody binding and development of a strong ELISA signal.
Supernatants of nonstimulated neutrophils gave no signal, even if both antibodies were used, due to lack of NETs formed.
High doses of TNF induce apoptosis in human neutrophils. While PMA triggered a robust signal in both assays as expected, apoptotic neutrophil samples failed to increase absorbance significantly over the nonstimulated values.
The assays are very sensitive and remain close to saturated until the 1:64 dilutions step. The changes in the measured OD values reflect proportional changes in the amounts of released NETs.
Both laboratory standard strains induced NET release but PA14 was more efficient than PAO1. PA14 and PMA evoked comparable levels of NET release. The data measured by the two assays showed strong linear correlation.
The NADPH oxidase inhibitor, diphenylene iodonium (DPI) blocked superoxide production elicited by all stimuli. DPI significantly inhibited the release of MPO–DNA complexes from neutrophils activated by all three stimuli.
Nonstimulated cells did not release NETs. When neutrophils were stimulated with PA14, CF isolate 10,035 or 10,155, NETs were formed. Both, MPO and HNE co-localized with extracellular DNA in NETs elicited by all three Pseudomonas strains.
Washing the cells before collecting NETs produced unre- liable data in our hands, as it is difficult to control whether fragile NETs are removed or not when collecting culture supernatants.