Molecular Markers of Eosinophilopoiesis at Birth: Kinetics of Cord Blood GATA-1, MBP and IL-5 Receptor Expression

Abstract

RATIONALE: Using colony assays and flow cytometry, we have recently shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and infant clinical outcomes. The current study aimed to utilize real-time polymerase chain reaction (Q-PCR) to ascertain the kinetic patterns of expression of CB Eo/B-lineage specific genes, GATA-1, MBP and IL-5Rα in order to develop molecular markers of Eo/B differentiation. METHODS: CB non-adherent mononuclear cells (NAMNCs) were isolated from random fresh and frozen samples, and incubated in the presence of rhIL-5 (1 ng/mL). At 24, 48 and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP were determined utilizing comparative Q-PCR in a multiplex reaction. The relative expression ratios between stimulated and un-stimulated cells were calculated using the delta-delta Ct method. RESULTS: Stimulation with IL-5 resulted in an up-regulation of GATA-1 expression; this peaked between 24 and 48hrs. In contrast, MBP was up-regulated in a slowly progressive pattern, with maximal up-regulation at 72h, while there was a stable, minor down-regulation of IL-5Rα. In keeping with these molecular kinetic findings, Eo/B colony-forming cells, grown in 14-day methylcellulose culture, were found to be present in relation to timing of GATA-1 expression. CONCLUSIONS: Multiplex Q-PCR analysis of mRNA from CB mononuclear cells stimulated with IL-5 demonstrates sequential expression of critical lineage-specific events, and can be used as a surrogate, molecular marker of CB Eo/B differentiation in clinical studies.