Molecular markers of eosinophilopoiesis at birth: kinetics of cord blood GATA-1, MBP and IL-5 receptor expression

Ellis, A.K., Crawford, L. and Denburg, J.A. (2006) Molecular markers of eosinophilopoiesis at birth: kinetics of cord blood GATA-1, MBP and IL-5 receptor expression. Journal of Allergy and Clinical Immunology, 117(2), S60. (doi: 10.1016/j.jaci.2005.12.241)

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Publisher's URL: http://dx.doi.org/10.1016/j.jaci.2005.12.241

Abstract

<b>RATIONALE:</b> 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.<p></p> <b>METHODS:</b> 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.<p></p> <b>RESULTS:</b> 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.<p></p> <b>CONCLUSIONS:</b> 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.<p></p>

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Stewart, Ms Lynn
Authors: Ellis, A.K., Crawford, L., and Denburg, J.A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Allergy and Clinical Immunology
Journal Abbr.:J. Allergy Clin. Immunol.
Publisher:Elsevier
ISSN:0091-6749
ISSN (Online):1097-6825

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