The induction of eosinophil peroxidase release: improved methods of measurement and stimulation

The production and release of eosinophil peroxidase (EPO) has been associated with human pathology. Degranulation assays with eosinophils are typically very difficult to do, with very low release values. EPO is unique for its high cationic charge. As such, it adheres to most extracellular surfaces, rendering it more difficult to measure compared with other released cellular proteins. Based on the understanding of the sticky nature of EPO, we were concerned that EPO released in vitro cannot be reproducibly measured in the supernatants of stimulated cells. Instead, we suspected that much of the released EPO was left adherent to the tube walls. We chose to investigate the measurement of EPO activity using the peroxidase substrate, O-phenylenediamine (OPD). Unlike other peroxidase substrates, OPD is soluble in aqueous physiological solutions, which do not lyse cell membranes, thereby allowing us to add OPD directly to eosinophils and exclusively measure extracellular EPO. This novel approach would remove the concerns of incorrect EPO measurements due to its adhesive nature. In addition, we developed this method to quantify EPO release in terms of EPO concentration. Finally, using this technique, we have been able to demonstrate secretory IgA (s-IgA)-induced release of EPO. By using OPD, we have developed a more sensitive and specific method to analyze the release of extracellular EPO.