Immunofluorescence analysis of cytokine and granule protein expression during eosinophil maturation from cord blood–derived CD34+ progenitors

BACKGROUND: In allergic inflammation and asthma, eosinophils are major effector cells. They have been shown to synthesize at least 23 cytokines, some of which are stored intracellularly in their unique crystalloid granules together with cationic granule protein. Little is known about the synthesis and storage of cytokines relative to cationic granule proteins in maturing eosinophils during eosinophilopoiesis. OBJECTIVE: Our purpose was to analyze the expression of eosinophil-derived mediators, major basic protein (MBP), eosinophil cationic protein (ECP), IL-6, and RANTES, during early stages of eosinophil maturation in CD34(+) cell-derived colonies. METHODS: Purified human cord blood CD34(+) cells were grown in methylcellulose cultures in the presence of recombinant human IL-3 and IL-5. By confocal laser scanning microscopy, the coexpression of eosinophil granular proteins MBP and ECP was determined concurrently with IL-6 and RANTES during eosinophil maturation on days 16, 19, 23, and 28 of culture. RESULTS: Immunoreactivity against MBP, ECP, IL-6, and RANTES was not detectable in freshly purified CD34(+) cells. Maturing eosinophils (>95%) exhibited positive immunostaining for all these proteins between days 16 and 28 of culture. At early stages of culture, discrete immunostaining was observed around the periphery but not in the center of granular structures. By day 28 cultured eosinophil-like cells showed evidence of the acquisition of crystalloid granule-like structures, analogous to those observed in mature peripheral blood eosinophils. CONCLUSIONS: Eosinophils express and store cytokines simultaneously with cationic granule proteins during the process of maturation. We propose that the storage of cytokines during the development of eosinophils is an early event and it may be integral to inflammatory responses involving these cells. The results of this study suggest a potential immunoregulatory function for maturing eosinophils.