Spi-C Negatively Regulates Eosinophil Peroxidase Production in Murine Eosinophils

Indianapolis, IN

Eosinophils are granular white blood cells that are important components of immune responses through the release of their granules like eosinophil peroxidase (EPO) upon activation. The release of this enzyme due to lysis or activation can be quantified with a colorimetric EPO assay. The assay can identify regulators of EPO expression and release, such as Spi-C, a transcription factor that is hypothesized to regulate expression of granule proteins in eosinophils. We hypothesize that Spi-Cko cells produce and release more EPO upon activation. Murine low density bone marrow was cultured for 8, 11, or 14 days in the presence of 10 ng/mL interleukin-5 (IL-5); resuspended in Roswell Memorial Park Institute (RPMI) media with 0.5% BSA; and treated for 2 hours with activators or lysis reagents. An EPO assay was performed and the color change was read at 492 nm. Phorbol myristate acetate (PMA) at concentrations of 100 ng/mL and 500 ng/mL induced dose-dependent release of EPO through activation of the cells plated at an optimal concentration of 1000 cells/µL. Relative to wild-type cells, Spi-Cko cells upon lysis released greater EPO at all of the time points of culture. At day 8 of maturation, Spi-Cko cells released more EPO than wild-type cells upon activation with 100 ng/mL PMA. These findings confirm that Spi-C negatively regulates the production and release of EPO in eosinophils, as its absence leads to greater release after activation or lysis. Future directions will involve further analysis of the role of Spi-C in the expression of eosinophilic granular proteins.