Cord blood CD34 + hemopoietic progenitor eosinophilic lineage commitment assessed by Q-PCR

Ellis, A.K., Crawford, L. and Denburg, J.A. (2009) Cord blood CD34 + hemopoietic progenitor eosinophilic lineage commitment assessed by Q-PCR. Journal of Allergy and Clinical Immunology, 123(2), S225. (doi: 10.1016/j.jaci.2008.12.862)

Abstract

RATIONALE: We have recently demonstrated multiplex Q-PCR analyses of mRNA expression of key eosinophil-lineage commitment events in cord blood (CB) non-adherent mononuclear cells (NAMNCs) after stimulation with IL-5. We hypothesized that the majority of these events were occurring in the differentiating pluripotent CD34+ cell population. The objective was to ascertain the kinetic patterns of expression of CB eosinophil-lineage specific genes, namely GATA-1, MBP, and IL-5Rα, in a purified CB CD34+ fraction.<p></p> METHODS: CB mononuclear cells were isolated from random fresh and frozen samples, separated by magnetic cell-sorting into a highly- purified CD34+ population, and then incubated in the presence of 1 ng/mL rhIL-5. At various time-points post-stimulation, RNA was isolated, reverse transcribed, and expression of GATA-1, IL-5Rα, and MBP mRNA determined, utilizing multiplex Q-PCR. Relative expression ratios of stimulated and un-stimulated cells were calculated using the ΔΔCt method.<p></p> RESULTS: Stimulation of CD34+ cells with IL-5 resulted in up-regulation of GATA-1 mRNA, peaking between 24 and 48 h, an identical pattern to that seen with NAMNCs (r = 0.963); MBP mRNA was up-regulated progressively, maximally at 96 h correlation with NAMNC, r = 0.978). Total IL-5R mRNA was stable at all time-points, with differential expression of mRNA for IL-5R soluble (Sol-) and transmembrane (Tm-) isoforms: At 24 h, the ratio of Tm-IL-5Rα to Sol-IL5Rα was 1.5:1, but this substantially reversed by 72 h, with a Sol-IL-5Rα to Tm-IL-5Rα ratio of 9.4:1.<p></p> CONCLUSIONS: Multiplex Q-PCR analyses of mRNA from CB CD34+ cells stimulated with IL-5 demonstrate sequential expression of critical eosinophil lineage-specific events, providing potential surrogate molecular markers of CB eosinophil differentiation.<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 > School of Infection & Immunity
• Journal Name: Journal of Allergy and Clinical Immunology
• Journal Abbr.: J. Allergy Clin. Immunol.
• Publisher: Mosby, Inc.
• ISSN: 0091-6749
• ISSN (Online): 1097-6825