Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA

Stéphane Esnault, James S. Malter

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV- driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t(1/2) in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t(1/2) of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded; suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM- CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow derailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.

Original languageEnglish (US)
Pages (from-to)5228-5234
Number of pages7
JournalJournal of Immunology
Volume163
Issue number10
StatePublished - Nov 15 1999

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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