Neutrophil granule proteins generate bactericidal ammonia chloramine on reaction with hydrogen peroxide.

The neutrophil enzyme, myeloperoxidase, by converting hydrogen peroxide (H2O2) and chloride to hypochlorous acid (HOCl), provides important defense against ingested micro-organisms. However, there is debate about how efficiently HOCl is produced within the phagosome and whether its reactions with phagosomal constituents influence the killing mechanism. The phagosome is a small space surrounding the ingested organism, into which superoxide, H2O2 and high concentrations of proteins from cytoplasmic granules are released. Previous studies imply that HOCl is produced in the phagosome, but a large proportion should react with proteins before reaching the microbe. To mimic these conditions, we subjected neutrophil granule extract to sequential doses of H2O2. Myeloperoxidase in the extract converted all the H2O2 to HOCl, which reacted with the granule proteins. 3-Chlorotyrosine, protein carbonyls and large amounts of chloramines were produced. At higher doses of H2O2, the extract developed potent bactericidal activity against Staphylococcus aureus. This activity was due to ammonia monochloramine, formed as a secondary product from protein chloramines and dichloramines. Isolated myeloperoxidase and elastase also became bactericidal when modified with HOCl and antibacterial activity was seen with a range of species. Comparison of levels of protein modification in the extract and in phagosomes implies that a relatively low proportion of phagosomal H2O2 would be converted to HOCl, but there should be sufficient for substantial protein chloramine formation and some breakdown to ammonia monochloramine. It is possible that HOCl could kill ingested bacteria by an indirect mechanism involving protein oxidation and monochloramine formation.

Protein chlorination in neutrophil phagosomes and correlation with bacterial killing.

Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.

Analysis of neutrophil bactericidal activity.

This chapter describes two methods for measuring the bactericidal activity of neutrophils. These are a new simple fluorescence-based assay, which quantifies bactericidal activity by measuring changes in bacterial fluorescence associated with a loss of membrane potential over time, and a more traditional colony counting protocol. Two variations of these techniques are presented: a "one-step" protocol providing a composite measure of phagocytosis and killing, and a "two-step" protocol that allows calculation of separate rate constants for both of these processes.

Analysis of neutrophil bactericidal activity.

The primary function of neutrophils is to engulf and destroy invading pathogens. If the bactericidal capacity of neutrophils is defective, an individual may suffer from enhanced susceptibility to potentially fatal microbial infection. To identify such defects, and to investigate the mechanisms used to kill bacteria, the bactericidal activity of neutrophils must be accurately quantified. This chapter provides details of a comprehensive microbiological technique that quantifies neutrophil bactericidal activity by measuring the loss of viability of ingested bacteria over time. Two variations of this technique are presented: a simple "one-step" protocol providing a composite measure of phagocytosis and killing, and a more advanced "two-step" protocol that allows calculation of separate rate constants for both of these processes.