Interleukin-4 and RANTES expression in maturing eosinophils derived from human cord blood CD34(+) progenitors

Eosinophils elaborate a number of proinflammatory mediators, including immu noregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are im portant cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occ urs in nascent eosinophils, leading to storage of newly produced proteins i n crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from pur ified cord blood CD34(+) cells and generated in semisolid media (methylcell ulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine m RNA profiles were analysed by the reverse transcription-polymerase chain re action (RT-PCR) to determine transcription of IL-4 and RANTES in cells on d ays 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine p roducts and granule major basic protein (MBP) was confirmed, from day 23 on wards, for colonies cultured in semisolid media, by immunofluorescent label ling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequ ences encoding IL-4 and RANTES were expressed in freshly prepared, non-diff erentiated CD34(+) cells. Furthermore, RANTES mRNA localized to carbol chro motrope 2R-positive colony cells, as assessed using in situ RT-PCR on day 2 1 of culture in semisolid media, and was found to gradually decrease (relat ive to beta2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (com prising > 90% eosinophil-like cells) up to day 28. Immunoreactivity for IL- 4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest th at synthesis and storage of immunoregulatory cytokines, essential for proce sses associated with adaptive immunity, occurs in nascent eosinophils durin g their growth and differentiation.