Transcriptional rates of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-3, and macrophage colony-stimulating factor genes in activated cord versus adult mononuclear cells: alteration in cytokine expression may be secondary to posttranscriptional instability
Lee, S.M.; Knoppel, E.; van de Ven, C.; Cairo, M.S.
Pediatric Research 34(5): 560-564
ISSN/ISBN: 0031-3998 PMID: 7506824 DOI: 10.1203/00006450-199311000-00002
We have previously demonstrated that protein production and mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and IL-3 are decreased in activated mononuclear cells (MNC) from human umbilical cord compared with adult peripheral blood. Reduced production of these colony-stimulating factors (CSF) during states of increased demand, as occurs during overwhelming bacterial infection, may play a role in the pathogenesis of neutropenia and thrombocytopenia in the newborn. To determine whether the reduced mRNA expression and CSF production from activated cord MNC is secondary to the decreased transcriptional activity of the corresponding genes, we determined the transcriptional rate of GM-CSF, G-CSF, IL-3, and M-CSF by nuclear run-on assays. Cord and adult MNC were isolated by Ficoll-Hypaque density centrifugation. A total of 10(8) MNC from cord and adult blood were stimulated as follows: GM-CSF and G-CSF [32 nmol/L phorbol-12-myristate-6-acetate (20 micrograms/L) + 2 mg/L phytohemagglutinin for 6 h]; IL-3 [32 nmol/L phorbol-12-myristate-6-acetate (20 micrograms/L) + 0.5 mumol/L A 23187 for 6 h]; and macrophage CSF (2 micrograms/L recombinant human GM-CSF for 24 h). The nuclei from unstimulated and stimulated cells were isolated and labeled with 32P-uridine triphosphate. Newly elongated 32P-labeled RNA transcripts were hybridized to slot blots of CSF DNA. To minimize cross hybridization artifacts, short fragments (0.5-1.0 kb) of cDNA were used.