scholarly journals Decreased macrophage colony-stimulating factor mRNA expression from activated cord versus adult mononuclear cells: altered posttranscriptional stability

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4269-4277 ◽  
Author(s):  
Y Suen ◽  
SM Lee ◽  
J Schreurs ◽  
E Knoppel ◽  
MS Cairo
Keyword(s):  
Mrna Expression ◽  
Protein Production ◽  
Mononuclear Cells ◽  
Human Umbilical Cord ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Mrna Induction ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

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)

scholarly journals Stimulation of nonclonal hematopoiesis and suppression of the neoplastic clone after treatment with recombinant human granulocyte- macrophage colony-stimulating factor in a patient with therapy-related myelodysplastic syndrome

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1491-1498 ◽  
Author(s):  
S Vadhan-Raj ◽  
HE Broxmeyer ◽  
G Spitzer ◽  
A LeMaistre ◽  
S Hultman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fragment Length Polymorphism ◽  
Marrow Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Selective Growth Advantage ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract A complete hematologic remission was achieved in a patient with therapy- related preleukemia and transfusion-dependent pancytopenia after treatment with recombinant human granulocyte-macrophage colony- stimulating factor (GM-CSF). The patient remained in remission for nearly 1 year despite the discontinuation of GM-CSF treatment. Several lines of evidence suggest that normal hematopoiesis was restored after GM-CSF treatment. First, the cytogenetic anomaly, which was present before GM-CSF, completely disappeared after three cycles of treatment. Cytogenetic conversion was documented by conventional karyotypic evaluation of mitotic bone marrow cell preparations as well as by premature chromosome condensation analysis of the nonmitotic cells of bone marrow and peripheral blood. Second, the growth pattern and cycle status of bone marrow granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells were found to be normal during remission. Third, X chromosome-linked restriction fragment length polymorphism- methylation analysis of DNA from mononuclear cells (greater than 80% lymphocytes) and mature myeloid elements showed a polyclonal pattern. These findings suggest that restoration of hematopoiesis in this patient after GM-CSF treatment may have resulted from suppression of the abnormal clone and a selective growth advantage of normal elements.

scholarly journals Stimulation of nonclonal hematopoiesis and suppression of the neoplastic clone after treatment with recombinant human granulocyte- macrophage colony-stimulating factor in a patient with therapy-related myelodysplastic syndrome

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1491-1498 ◽  
Author(s):  
S Vadhan-Raj ◽  
HE Broxmeyer ◽  
G Spitzer ◽  
A LeMaistre ◽  
S Hultman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fragment Length Polymorphism ◽  
Marrow Cell ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Selective Growth Advantage ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

A complete hematologic remission was achieved in a patient with therapy- related preleukemia and transfusion-dependent pancytopenia after treatment with recombinant human granulocyte-macrophage colony- stimulating factor (GM-CSF). The patient remained in remission for nearly 1 year despite the discontinuation of GM-CSF treatment. Several lines of evidence suggest that normal hematopoiesis was restored after GM-CSF treatment. First, the cytogenetic anomaly, which was present before GM-CSF, completely disappeared after three cycles of treatment. Cytogenetic conversion was documented by conventional karyotypic evaluation of mitotic bone marrow cell preparations as well as by premature chromosome condensation analysis of the nonmitotic cells of bone marrow and peripheral blood. Second, the growth pattern and cycle status of bone marrow granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells were found to be normal during remission. Third, X chromosome-linked restriction fragment length polymorphism- methylation analysis of DNA from mononuclear cells (greater than 80% lymphocytes) and mature myeloid elements showed a polyclonal pattern. These findings suggest that restoration of hematopoiesis in this patient after GM-CSF treatment may have resulted from suppression of the abnormal clone and a selective growth advantage of normal elements.

scholarly journals Production of granulocyte-macrophage colony-stimulating factor by large granular lymphocytes stimulated with Candida albicans: role in activation of human neutrophil function

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2259-2265 ◽  
Author(s):  
DK Blanchard ◽  
MB Michelini-Norris ◽  
JY Djeu
Keyword(s):  
Candida Albicans ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Yeast Cells ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Granular Lymphocytes

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF-producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM- CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

scholarly journals Harvesting and enrichment of hematopoietic progenitor cells mobilized into the peripheral blood of normal donors by granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF: potential role in allogeneic marrow transplantation

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 275-282 ◽  
Author(s):  
TA Lane ◽  
P Law ◽  
M Maruyama ◽  
D Young ◽  
J Burgess ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Allogeneic Transplantation ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

To explore the use of stem/progenitor cells from peripheral blood (PB) for allogeneic transplantation, we have studied the mobilization of progenitor cells in normal donors by growth factors. Normal subjects were administered either granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 micrograms/kg/d, or G-CSF at 10 micrograms/kg/d, or a combination of G- and GM-CSF at 5 micrograms/kg/d each, administered subcutaneously for 4 days, followed by leukapheresis on day 5. Mononuclear cells expressing CD34 (CD34+ cells) were selectively enriched by affinity labeling using Dynal paramagnetic microspheres (Baxter Isolex; Baxter Healthcare Corp, Santa Ana, CA). The baseline CD34+ cells in peripheral blood before mobilization was 0.07% +/- 0.05% (1.6 +/- 0.7/microL; n = 18). On the fifth day after stimulation (24 hours after the fourth dose), the CD34+ cells were 0.99% +/- 0.40% (61 +/- 14/microL) for the 8 subjects treated with G-CSF, 0.25% +/- 0.25% (3 +/- 3/microL, both P < .01 v G-CSF) for the 5 subjects administered GM-CSF, and for the 5 subjects treated with G- and GM-CSF, 0.65% +/- 0.28% (41 +/- 18/microL, P < .5 v GM-CSF). Parallel to this increase in CD34+ cells, clonogenic assays showed a corresponding increase in CFU- GM and BFU-E. The total number of CD34+ cells collected from the G-CSF group during a 3-hour apheresis was 119 +/- 65 x 10(6) and was not significantly different from that collected from the group treated with G- and GM-CSF (101 +/- 35 x 10(6) cells), but both were greater than that from the group treated with GM-CSF (12.6 +/- 6.1 x 10(6); P < .01 for both comparisons). Analysis of the CD34+ subsets showed that a significantly higher percentage of cells with the CD34+/CD38- phenotype is found after mobilization with G- and GM-CSF. In the G-CSF group, immunomagnetic selection of CD34+ cells permitted the enrichment of the CD34+ cells in the apheresis product to 81% +/- 11%, with a 48% +/- 12% yield and to a purity of 77% +/- 21% with a 51% +/- 15% recovery in the G- and GM-CSF group. T cells were depleted from a mean of 4.5 +/- 2.0 x 10(9) to 4.3 +/- 5.2 x 10(6) after selection, representing 99.9% depletion. We conclude that it is feasible to collect sufficient numbers of PB progenitor cells from normal donors with one to two leukapheresis procedures for allogeneic transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Interferon-gamma increases the expression of the gene encoding the beta subunit of the granulocyte-macrophage colony-stimulating factor receptor

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1736-1742 ◽  
Author(s):  
M Hallek ◽  
EM Lepisto ◽  
KE Slattery ◽  
JD Griffin ◽  
TJ Ernst
Keyword(s):  
Mrna Expression ◽  
Interferon Gamma ◽  
De Novo ◽  
Beta Subunit ◽  
Colony Stimulating Factor ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high-affinity receptors (GM-CSF-R) consisting of at least two distinct subunits, GM-CSF-R alpha and GM-CSF-R beta. The genes of these GM-CSF-R subunits have been identified recently, but little is known about the regulation of their expression. In this study, we investigated the expression of the GM-CSF- R subunit genes in normal human monocytes. Out of a panel of various cytokines and factors tested, only interferon-gamma (IFN-gamma) affected the expression of one of the GM-CSF-R subunit genes by increasing the GM-CSF-R beta mRNA expression threefold to sixfold with no effect on GM-CSF-R alpha. Maximal effects occurred 2 to 4 hours after stimulation with 500 to 5,000 U/mL IFN-gamma. Nuclear run-on assays and mRNA half-life studies showed that IFN-gamma modestly enhanced the transcription of the GM-CSF-R beta gene and stabilized the GM-CSF-R beta mRNA, with the latter mechanism predominant. Pretreatment of the monocytes with cycloheximide did not abrogate the increase of GM- CSF-R beta mRNA expression induced by IFN-gamma, indicating that de novo protein synthesis was not required for this activity. When monocytes were exposed to IFN-gamma for 6 to 24 hours, the number of GM- CSF-R per cell was increased 79% as compared with controls, whereas the receptor affinity remained unchanged. These data indicate that the GM- CSF-R expression in monocytes may be upregulated by IFN-gamma via an increased expression of the beta subunit gene, involving both transcriptional and post-transcriptional mechanisms.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 mRNAs are produced by a small fraction of blood mononuclear cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1525-1530 ◽  
Author(s):  
JZ Wimperis ◽  
CM Niemeyer ◽  
CA Sieff ◽  
B Mathey-Prevot ◽  
DG Nathan ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Blood Mononuclear Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Northern blot analysis has identified granulocyte macrophage colony stimulating factor (GM-CSF) mRNA in monocytes and both GM-CSF and interleukin-3 (IL-3) mRNA in lymphocytes. However, these results have not addressed whether all cells or a subset of the population is capable of hematopoietic growth factor (HGF) production. To resolve this question, we applied in situ hybridization of radiolabeled antisense RNA probes to centrifuged preparations of total blood mononuclear cells (BMCs) and fractionated lymphocyte subpopulations. Without stimulation, no circulating cells expressed detectable levels of GM-CSF or IL-3 mRNA. On stimulation of BMCs with phorbol myristate acetate (PMA) and phytohemagglutinin or PMA and the calcium ionophore ionomycin, approximately 5% expressed GM-CSF mRNA and approximately 1% IL-3 mRNA. Control sense probes produced no labeled cells. To determine the subsets of lymphocytes capable of GM-CSF and IL-3 expression, BMCs were fractionated by FACS into CD8+ and CD4+ lymphocyte subsets and CD16+ (NK) cells. The unfractionated cells and cell fractions were then stimulated with PMA and ionomycin. Results demonstrated that 3% to 5% of the CD16+, CD8+, and CD4+ lymphocytes produced GM-CSF mRNA. However, the number of IL-3 mRNA-positive cells in the FACS-sorted subsets was greatly reduced (0.02% to 0.05%) as compared with the unseparated cells (1%). Treatment of BMCs with high-dose interleukin-2 (IL-2) for 1 week followed by PMA plus ionomycin resulted in a lymphocyte population in which 50% and 3% of cells expressed GM-CSF and IL-3 mRNA, respectively. Thus, GM-CSF and IL-3 mRNA expression in T cells and NK cells is restricted to a small fraction of cells that can be greatly expanded by IL-2 stimulation. These results suggest a possible physiologic mechanism for increasing HGF production by circulating lymphocytes.

scholarly journals Granulocyte-macrophage colony-stimulating factor and interleukin-3 mRNAs are produced by a small fraction of blood mononuclear cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1525-1530 ◽  
Author(s):  
JZ Wimperis ◽  
CM Niemeyer ◽  
CA Sieff ◽  
B Mathey-Prevot ◽  
DG Nathan ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Blood Mononuclear Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Northern blot analysis has identified granulocyte macrophage colony stimulating factor (GM-CSF) mRNA in monocytes and both GM-CSF and interleukin-3 (IL-3) mRNA in lymphocytes. However, these results have not addressed whether all cells or a subset of the population is capable of hematopoietic growth factor (HGF) production. To resolve this question, we applied in situ hybridization of radiolabeled antisense RNA probes to centrifuged preparations of total blood mononuclear cells (BMCs) and fractionated lymphocyte subpopulations. Without stimulation, no circulating cells expressed detectable levels of GM-CSF or IL-3 mRNA. On stimulation of BMCs with phorbol myristate acetate (PMA) and phytohemagglutinin or PMA and the calcium ionophore ionomycin, approximately 5% expressed GM-CSF mRNA and approximately 1% IL-3 mRNA. Control sense probes produced no labeled cells. To determine the subsets of lymphocytes capable of GM-CSF and IL-3 expression, BMCs were fractionated by FACS into CD8+ and CD4+ lymphocyte subsets and CD16+ (NK) cells. The unfractionated cells and cell fractions were then stimulated with PMA and ionomycin. Results demonstrated that 3% to 5% of the CD16+, CD8+, and CD4+ lymphocytes produced GM-CSF mRNA. However, the number of IL-3 mRNA-positive cells in the FACS-sorted subsets was greatly reduced (0.02% to 0.05%) as compared with the unseparated cells (1%). Treatment of BMCs with high-dose interleukin-2 (IL-2) for 1 week followed by PMA plus ionomycin resulted in a lymphocyte population in which 50% and 3% of cells expressed GM-CSF and IL-3 mRNA, respectively. Thus, GM-CSF and IL-3 mRNA expression in T cells and NK cells is restricted to a small fraction of cells that can be greatly expanded by IL-2 stimulation. These results suggest a possible physiologic mechanism for increasing HGF production by circulating lymphocytes.

scholarly journals Interferon-gamma increases the expression of the gene encoding the beta subunit of the granulocyte-macrophage colony-stimulating factor receptor

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1736-1742
Author(s):  
M Hallek ◽  
EM Lepisto ◽  
KE Slattery ◽  
JD Griffin ◽  
TJ Ernst
Keyword(s):  
Mrna Expression ◽  
Interferon Gamma ◽  
De Novo ◽  
Beta Subunit ◽  
Colony Stimulating Factor ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high-affinity receptors (GM-CSF-R) consisting of at least two distinct subunits, GM-CSF-R alpha and GM-CSF-R beta. The genes of these GM-CSF-R subunits have been identified recently, but little is known about the regulation of their expression. In this study, we investigated the expression of the GM-CSF- R subunit genes in normal human monocytes. Out of a panel of various cytokines and factors tested, only interferon-gamma (IFN-gamma) affected the expression of one of the GM-CSF-R subunit genes by increasing the GM-CSF-R beta mRNA expression threefold to sixfold with no effect on GM-CSF-R alpha. Maximal effects occurred 2 to 4 hours after stimulation with 500 to 5,000 U/mL IFN-gamma. Nuclear run-on assays and mRNA half-life studies showed that IFN-gamma modestly enhanced the transcription of the GM-CSF-R beta gene and stabilized the GM-CSF-R beta mRNA, with the latter mechanism predominant. Pretreatment of the monocytes with cycloheximide did not abrogate the increase of GM- CSF-R beta mRNA expression induced by IFN-gamma, indicating that de novo protein synthesis was not required for this activity. When monocytes were exposed to IFN-gamma for 6 to 24 hours, the number of GM- CSF-R per cell was increased 79% as compared with controls, whereas the receptor affinity remained unchanged. These data indicate that the GM- CSF-R expression in monocytes may be upregulated by IFN-gamma via an increased expression of the beta subunit gene, involving both transcriptional and post-transcriptional mechanisms.

scholarly journals Regulation of parasite-induced eosinophilia: selectively increased interleukin 5 production in helminth-infected patients.

1990 ◽  
Vol 172 (1) ◽  
pp. 399-402 ◽  
Author(s):  
A P Limaye ◽  
J S Abrams ◽  
J E Silver ◽  
E A Ottesen ◽  
T B Nutman
Keyword(s):  
Mononuclear Cells ◽  
Mrna Level ◽  
Colony Stimulating Factor ◽  
Peripheral Blood Mononuclear ◽  
Interleukin 5 ◽  
Gm Csf ◽  
Helminth Infections ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Production of the eosinophilogenic cytokines interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5 by mitogen-stimulated peripheral blood mononuclear cells was compared between 11 noneosinophilic individuals and seven patients with helminth-induced eosinophilia. Both the kinetics and quantities of IL-3 and GM-CSF were similar in the two groups. In contrast, IL-5 production at both the protein and the mRNA level was markedly greater in the eosinophilic patients, an observation suggesting that IL-5 may be particularly important in mediating the selective eosinophilia seen in filarial and other helminth infections.

scholarly journals Increased granulocyte-macrophage colony-stimulating factor mRNA instability in cord versus adult mononuclear cells is translation- dependent and associated with increased levels of A + U-rich element binding factor

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2889-2897 ◽  
Author(s):  
JS Buzby ◽  
SM Lee ◽  
P Van Winkle ◽  
CT DeMaria ◽  
G Brewer ◽  
...  
Keyword(s):  
Half Life ◽  
Mononuclear Cells ◽  
Protein Isoforms ◽  
Colony Stimulating Factor ◽  
Mobility Shift ◽  
Gm Csf ◽  
Binding Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is fourfold lower in phorbol myristate acetate (PMA) + phytohemagglutinin (PHA)-activated mononuclear cells (MNC) from newborns compared with adults. The GM-CSF transcription rate is similar in umbilical cord and adult MNC, but transcript half-life is threefold lower in cord activated MNC. Interaction of RNA binding proteins, such as the cloned adenosine + uridine-rich element, binding factor, AUF1, with eight AUUUA motifs in the human GM-CSF mRNA 3′-untranslated region (GM-3′-UTR) has been implicated in regulating transcript stability. Translational inhibition by cycloheximide (CHX) significantly increased GM-CSF mRNA accumulation and half-life by three-fold in activated cord MNC, but had a minimal effect in activated adult MNC as compared with PMA + PHA alone. Electrophoretic mobility-shift assays with a 32P-labeled, 305-nucleotide RNA comprising the GM-3′-UTR revealed two RNaseT1-resistant, bound complexes that were almost twice as abundant in cord than in adult MNC extracts. Mobility-shift competition assays and RNaseT1 mapping localized the binding site of both complexes to a 52-nucleotide region containing seven of eight AUUUA motifs. Inclusion of AUF1 antiserum produced a supershifted complex at 35-fold higher levels in cord than in adult MNC extracts. Extracts from the carcinoma cell line 5637, with extended GM-CSF mRNA half-life, also had very low levels of anti-AUF1 supershifted complex. Anti-AUF1 immunoblotting showed significantly higher levels of two AUF1 protein isoforms and lower levels of one in cord than in adult MNC or 5637 extracts. These results suggest that destabilization of GM-CSF mRNA in cord MNC is translation-dependent and that increased levels of specific AUF1 isoforms in cord MNC may target transcripts for increased degradation, which could account in part for dysregulation of neonatal phagocytic immunity.

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