Abstract
The neutrophil is the most abundant human leukocyte and is essential to innate immunity. Neutrophils kill bacteria inside phagosomal compartments using a combination of toxic oxidants, proteases and cationic peptides. Surprisingly, there is still only little understanding of how ingested bacteria die. Upon phagocytosis of bacteria, the respiratory burst of neutrophils rapidly generates superoxide via the NADPH oxidase, feeding a complex chain of redox events. It is known that the neutrophil enzyme myeloperoxidase uses superoxide to produce microbicidal hypohalous acids. We aimed to measure intracellular hypochlorous acid (HOCl) to understand the role of this oxidant during normal phagocytosis. Recent development of oxidant specific probes and advances in cell monitoring techniques have aided the study of oxidant production by neutrophils. Neutrophils isolated from peripheral blood were incubated with opsonised zymosan particles, and a fluorescent probe, R19-S, was used to monitor phagosomal HOCl. The development of a sensitive flow cytometry method to quantify HOCl production inside individual cells has provided insight into the range and heterogeneity within an individual’s neutrophils. We have shown that this method can detect deficiencies in oxidant production by abnormal neutrophils. The potential for this method to aid in the diagnosis of clinical disorders was demonstrated in a patient with putative Chediak Higashi syndrome. Decreased capacity to generate HOCl in phagosomes was measured more easily than testing neutrophils for bacterial killing efficiency. The R19-S assay provides direct analysis of phagosomal activity. It could be a useful tool for understanding rare immunological disorders that are impacted by abnormal neutrophil function.