Transcriptional Rates of Granulocyte-Macrophage Colony-Stimulating Factor, Granulocyte Colony-Stimulating Factor, Interleukin-3, and Macrophage Colony-Stimulating Factor Genes in Activated Cord : Alteration in Cytokine Expression May Be Secondary to Posttranscriptional InstabilityVersus: Alteration in Cytokine Expression May Be Secondary to Posttranscriptional Instability Adult Mononuclear Cells: Alteration in Cytokine Expression May Be Secondary to Posttranscriptional Instability

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Abstract

ABSTRACT

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 ncutropenia 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 108 MNC from cord and adult blood were stimulated as follows: GM-CSF and G-CSF [32 nmol/L phorbol-12-myristate-6-acetate (20 μg/L) + 2 mg/L phytohemagglutinin for 6 h]; IL-3 [32 nmol/L phorbol-12-myristate-6-acctate (20 μg/L) + 0.5 μmol/L A 23187 for 6 h]; and macrophage CSF (2 μg/ 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. The transcriptional rate increase of GM-CSF, G-CSF, IL-3, and macrophage CSF upon stimulation appears to be similar in both cord and adult MNC [GM-CSF: 260 ± 62% (cord) versus 270 ± 33% (adult); G-CSF: 220 ± 71 % (cord) versus 220 ± 44% (adult); IL-3: 150 ± 25% (cord) versus 160 ± 38% (adult); macrophage CSF: 130 ± 10% (cord) versus 150 ± 15% (adult), mean ± SD]. These findings indicate that cord MNC transcribe these CSF genes at the same level as adult MNC during states of increased demand (stimulation). Therefore, the decrease in CSF“ mRNA expression in activated cord versus adult MNC is probably not secondary to defects in transcriptional regulation. Alteration in posttranscriptional events, such as dysregulation of mRNA stability, could account for the difference between newborn and adult CSF expression.

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