Effect of Interleukin–3, Interleukin 5 and Hyaluronic Acid on Cultured Eosinophils Derived from Human Umbilical Cord Blood Mononuclear Cells

Abstract

Background: Several studies have shown that cultured eosinophils can be generated from human umbilical cord blood mononuclear cells (UCMC) in the presence of interleukin (IL)–3 and IL–5 in vitro. Other reports have indicated that cellular adhesion to hyaluronic acid (HA) enhances the proliferation of cultured eosinophils derived from CD34+ cells purified from UCMC. The aim of this study was to obtain large numbers of mature eosinophils from UCMC using IL–3, IL–5 and HA, and to investigate their functions. Methods: We examined several combinations of IL–3 and IL–5 and their effect on eosinophil development from UCMC in HA–coated on non–coated flasks. We also examined whether cultured eosinophils degranulated eosinophil–derived neurotoxin (EDN) induced by secretory immunoglobulin A conjugated to sepharose beads (sIgA–beads) and responded to eotaxin. Results: Culture with HA–coated flasks for 35 days (in the presence of IL–3 and IL–5, with IL–3 omitted after day 14 of culture) caused a 11.2–fold augmentation in the proliferation of UCMC. On day 35 of the culture, 98% of cultured cells were eosinophils judging from May–Grünwald and Giemsa staining and transmission electron micrographs. The EDN content of the cultured eosinophils on day 35 was 156 ng/105 cells. Cultured eosinophils degranulated EDN induced by sIgA–beads and responded to eotaxin by chemotaxis and intracellular Ca2+ mobilization. Conclusion: We found a useful culture system to obtain large numbers of eosinophils derived from UCMC, which may facilitate the investigation of eosinophil function, since there was no significant difference in response to sIgA–beads and eotaxin between cultured and peripheral eosinophils.

Peripheral blood eosinophils from patients with allergic asthma contain increased intracellular eosinophil-derived neurotoxin

“…Even after 35 to 42 days of culture, when >95% of the cells expressed characteristic eosinophil morphology, the intracellular concentrations of EDN were lower (700-1600 ng/1 3 10 6 cells) than the concentrations observed in any of our groups. 23 Further culture of the developing cells resulted in a profound loss of EDN content. In contrast, Al-Rabia et al 24 reported expression of major basic protein and ECP by 15% to 20% of cultured peripheral blood CD34 + stem cells incubated with stem cell factor, FLT1 ligand, IL-3, and IL-5 for 3 to 7 days.…”

A Novel CC-Chemokine Receptor 3 Antagonist, Ki19003, Inhibits Airway Eosinophilia and Subepithelial/Peribronchial Fibrosis Induced by Repeated Antigen Challenge in Mice

“…The chemotaxis assay was performed using a 24-well microchemotaxis chamber as described previously with minor modifications (26). In brief, chemoattractants were diluted in RPMI 1640 medium supplemented with 1 mg/ ml BSA and placed in the lower wells (250 μ l/well).…”

Purification of functional eosinophils from human bone marrow

“…Dyer et al (2008), alternatively, described a series of culture conditions in which the addition of stem cell factor (SCF), FLT3-ligand, and IL-5 induced differentiation of mouse bone marrow progenitor cells into eosinophils. Human progenitor cells from the umbilical cord (Ten et al, 1991; Ohashi et al, 1999) and bone marrow (Clutterbuck et al, 1989) have also been successfully differentiated into eosinophils in vitro in the presence of IL-3 and IL-5. While these are viable options to obtain human bone marrow-derived eosinophils, it may not represent the best option as the differentiation of these cells from progenitor populations can be labor and/or time intensive (2–4 weeks) as well as costly.…”