Modulation of growth and differentiation of eosinophils from human peripheral blood CD34 + cells by IL5 and other growth factors

Abstract

Small numbers of CD34 + primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34 + cells. CD34 + cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34 + cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3 + GM-CSF + IL5), the addition of interferon-α or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-γ, tumor necrosis factor-α, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34 + cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10 4 cells plated at Day 3 and 134 colonies/10 4 cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34 + cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34 + cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts.