Molecular Markers of Eosinophilopoiesis: Multiplex Q-PCR Analysis of GATA-1, MBP and IL-5 Receptor mRNA Expression in Peripheral Blood

Abstract

RATIONALE: Using colony assays and flow cytometry, we have shown that eosinophil/basophil (Eo/B) progenitor phenotype and function are associated with atopic risk at birth and early childhood clinical outcomes. We have also recently demonstrated that real-time polymerase chain reaction (Q-PCR) can reveal kinetic patterns of expression in cord blood (CB) of several Eo/B lineage-specific genes, specifically GATA-1, MBP and IL-5Rα, as surrogate molecular markers of Eo/B differentiation. These same methods have yet to be established in peripheral blood (PB) samples. Our objective was to determine the kinetic patterns of expression of CB Eo/B-lineage specific genes in PB. METHODS: PB non-adherent mononuclear cells (PB NAMNC) were isolated from random fresh samples, and incubated in the presence of IL-5. At 24, 48, and 72h post-stimulation, RNA was isolated, reverse transcribed, and expression of IL-5Rα, GATA-1, and MBP was determined utilizing multiplex Q-PCR. Relative expression ratios of stimulated to un-stimulated cells were calculated using the delta-delta Ct method. RESULTS: Stimulation of PB NANMC with IL-5 resulted in an up-regulation of GATA-1 expression, peaking at 24h, with a slower return to baseline expression than that observed in CB. MBP expression was minimally altered at all time points, compared to CB, where slow up-regulation, maximal at 72h, had been observed. There was completely stable expression IL-5Rα, similar to that seen in CB. CONCLUSIONS: Multiplex Q-PCR analysis of mRNA from PB demonstrates expression of critical Eo/B lineage-specific events. Further investigation of the validity and utility of Q-PCR analyses of PB for surrogate, molecular markers Eo/B differentiation is underway.