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Decreased macrophage colony-stimulating factor mRNA expression from activated cord versus adult mononuclear cells:
1Division of Hematology/Oncology and Bone Marrow Transplantation, Children's Hospital of Orange County, CA 92668.
Abstract:
We have previously shown that protein production and mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin-3 are decreased in stimulated mononuclear cells (MNCs) from human umbilical cord compared with adult peripheral blood. These deficiencies may contribute to the increased susceptibility of neonates to infection. Macrophage colony-stimulating factor (M-CSF) regulates the proliferation, differentiation, and functional activation of monocytes. In the present study, we compared the regulation of M-CSF gene expression and protein production from stimulated cord and adult MNCs. Upon adhesion to tissue culture flasks, both cord and adult MNCs constitutively expressed M-CSF mRNA. In response to both adhesion and recombinant human GM-CSF (rhGM-CSF) stimulation for 120 hours, radioimmunoassays and bioassays showed that cord MNCs produced twofold to threefold less M-CSF protein compared with adult MNCs. Northern blot analysis also showed a fourfold decrease in M-CSF mRNA expression in both unstimulated and GM-CSF-induced cord versus adult MNCs. M-CSF mRNA expression in both cord and adult MNCs peaked between 16 and 24 hours and decreased to normal levels by 48 hours. We next determined the relative rates of transcription of the M-CSF gene by nuclear run-on assays in both cord and adult MNCs. The basal level signal of the M-CSF gene was similar between cord and adult MNCs. The transcriptional rate after stimulation with rhGM-CSF appeared to increase to a similar extent in both cord and adult MNCs (130% +/- 10% v 150% +/- 15%, C v A, n = 3, mean +/- SD). The comparative stability of M-CSF mRNA from cord versus adult MNCs was next determined by actinomycin D decay studies. The half-life of M-CSF mRNA from stimulated adult MNCs was 70 +/- 7.0 minutes (n = 4) compared with 47 +/- 2.8 minutes (n = 3) from stimulated cord MNCs (mean +/- SD, P < .05). To further determine the involvement of labile protein factors in posttranscriptional regulation, cord and adult MNCs were incubated with cycloheximide (CHX; 10 micrograms/mL). There was a significant increase in the induction of M-CSF mRNA by CHX treatment in both cord and adult MNCs. The increase of M-CSF mRNA induction by CHX was 2.5 times higher in cord MNCs compared with that in adult MNCs. These results suggest that there are one or more labile proteins that regulate M-CSF transcript stability in both cord and adult MNCs.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Neonatal mononuclear cells (MNCs) produce significantly less macrophage colony-stimulating factor (M-CSF) protein and mRNA compared to adult MNCs, potentially explaining increased infant infection susceptibility.
Area of Science:
- Immunology
- Cell Biology
- Neonatal Research
Background:
- Neonates exhibit increased susceptibility to infections, partly due to deficiencies in immune responses.
- Previous studies indicated reduced production of certain cytokines like GM-CSF, G-CSF, and IL-3 in neonatal cells.
- Macrophage colony-stimulating factor (M-CSF) is crucial for monocyte/macrophage development and function.
Purpose of the Study:
- To compare the regulation of M-CSF gene expression and protein production between neonatal (cord) and adult mononuclear cells (MNCs).
- To investigate the mechanisms underlying potential differences in M-CSF production, focusing on transcriptional and post-transcriptional regulation.
Main Methods:
- Mononuclear cells (MNCs) from human umbilical cord blood and adult peripheral blood were cultured and stimulated.
- M-CSF protein levels were quantified using radioimmunoassays and bioassays.
- M-CSF mRNA expression was analyzed via Northern blot and nuclear run-on assays.
- M-CSF mRNA stability was assessed using actinomycin D decay studies.
- The role of labile protein factors was investigated using cycloheximide (CHX) treatment.
Main Results:
- Cord MNCs produced 2-3 times less M-CSF protein and showed a 4-fold decrease in M-CSF mRNA expression compared to adult MNCs.
- Transcriptional rates of the M-CSF gene were similar between cord and adult MNCs upon stimulation.
- M-CSF mRNA from cord MNCs exhibited significantly lower stability (shorter half-life) than that from adult MNCs.
- Cycloheximide treatment induced M-CSF mRNA expression more significantly in cord MNCs than in adult MNCs, suggesting involvement of labile inhibitory proteins.
Conclusions:
- Neonatal mononuclear cells have impaired M-CSF production compared to adult cells, primarily due to post-transcriptional mechanisms.
- Reduced M-CSF mRNA stability and the action of labile inhibitory proteins contribute to lower M-CSF levels in neonates.
- These findings highlight a potential cellular mechanism underlying the heightened infection risk in newborns.