The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction.
In human long-term marrow cultures granulomonopoiesis is maintained for several weeks. Studies on granulomonocytic progenitors (CFU-GM) and their progeny have shown that survival, proliferation, differentiation and maturation of these cells are controlled by a set of glycoproteins, the colony-stimulating factors (CSFs) and the Steel factor. We have studied the expression of these factors using reverse transcriptase polymerase chain reaction (RT-PCR) in 17 adherent layers of normal bone marrow at 3, 5 or 7 weeks of culture. We have taken the 5637 bladder carcinoma cell line as a control for expression of GM-CSF, M-CSF, G-CSF and Steel factor, and PHA-activated T lymphocytes as a control for expression of multi-CSF (interleukin 3, IL-3). We have found that GM-CSF was expressed in the 17 adherent layers without induction by interleukin 1 beta (IL-1 beta). M-CSF was also detected in all cases, but in two early-stage (week 3 and week 5) cultures only after stimulation by IL-1 beta. G-CSF was detected in only 11 cases (three without IL-1 beta, and eight after addition of IL-1 beta). Steel factor was detected in 14 cases (ten without IL-1 beta, and four after addition of IL-1 beta). IL-3 was not detected even by means of nested RT-PCR. These data indicate in six late-stage (week 5 or week 7) cultures G-CSF messenger concentrations 10(3)-fold less than in 5637 control cells (for an identical amount of total cellular RNA). A similar conclusion may be drawn for Steel factor in three late-stage cultures. For IL-3 our negative results indicate a messenger concentration 10(5)-fold less than in activated T lymphocytes. These results suggest a crucial role for GM-CSF and M-CSF in the maintenance of granulomonopoiesis in human long-term cultures. The role of G-CSF and Steel factor may be more marginal. Eventually IL-2 may not be involved in the regulatory process.
Deschaseaux
M L
ML
Centre Regional de Transfusion Sanguine, Besançon, France.
Hervé
P
P
Charbord
P
P
D000199:Actins / Q000032:analysis; D001483:Base Sequence; D001853:Bone Marrow / Q000737:chemistry*; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000032:analysis*; D016179:Granulocyte Colony-Stimulating Factor / Q000032:analysis; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis; D016298:Hematopoietic Cell Growth Factors / Q000032:analysis*; D006801:Humans; D007377:Interleukin-3 / Q000032:analysis; D016173:Macrophage Colony-Stimulating Factor / Q000032:analysis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D019089:Stem Cell Factor; D014158:Transcription, Genetic
D000199:Actins; D003115:Colony-Stimulating Factors; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D012313:RNA; D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor