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

Abstract

SUMMARYEosinophils elaborate a number of proin¯ammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from puri®ed cord blood CD34 + cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA pro®les were analysed by the reverse transcription±polymerase chain reaction (RT±PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was con®rmed, from day 23 onwards, for colonies cultured in semisolid media, by immuno¯uorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, nondifferentiated CD34 + cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R-positive colony cells, as assessed using in situ RT±PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to b2-microglobulin) in rhIL-3-and rhIL-5-treated colony cells (comprising >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 that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation.

Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood–derived CD34+ cells

“…However, they only distinguished between blast cells and mature eosinophils without mentioning the other stages of eosinophil differentiation. The same percentages of cells that resembled eosinophils, after 21 days of differentiation, were described by Velazquez et al 50 Our differentiation protocol resulted in approximately the same percentages of differentiated and juvenile eosinophils as these previous studies. This ex vivo system of eosinophil differentiation resulted in a high number of juvenile eosinophils that were positive for LFB and eosinophil peroxidase (data not shown), and it is an important tool for analyzing the role of transcription factors during early stages of the differentiation process.…”

Mechanisms of eosinophil recruitment and activation

“…These mice had continued eosinophil presence in the bone marrow, suggesting that an additional role for IL-5 and GM-CSF may be mobilization of eosinophils from bone marrow to blood [13]. Other potentially eosinophilopoietic signals also are produced by Eo/B progenitors, including IL-4, IL-6, and RANTES (regulated upon activation, normal T cell expressed and secreted) [14,15]. Thus, in an autocrine fashion, eosinophils may self-induce their maturation and prolong their survival.…”

Eosinophil-Derived IL-10 Supports Chronic Nematode Infection

“…In mouse models, eosinophils influence insulin resistance (3, 4), promote the regenerative response to toxic injury of skeletal muscle (5) and liver (6), and are required for recruitment of Th2 cells to the lung in allergy (7–9). Eosinophils constitutively express IL-4 (10) and production of IL-4 or IL-13 is key to the role of eosinophils in each of these contexts. Eosinophils also regulate adaptive immunity by producing cytokines (11), and this property has been tested in experiments that are relevant to the outcome of worm infection.…”