Identification of the hematopoietic growth factors elaborated by bone marrow stromal cells using antibody neutralization analysis.

These studies were undertaken to characterize the subclasses of hematopoietic growth factors produced by stromal cells in long-term murine bone marrow cultures. Exposure of these cultures to extremely high doses of irradiation (500 Gy), followed by endotoxin stimulation, permitted detection and characterization of various growth factor activities in the unconcentrated conditioned medium. To determine the nature of these activities, neutralization studies were performed using antisera against the following subclasses of purified colony-stimulating factors (CSFs): purified L-cell CSF-1, recombinant granulocyte-macrophage CSF (rGM-CSF), and recombinant interleukin 3 (rIL3). The antiserum against CSF-1 consistently abrogated 100% of the CSF bioactivity in irradiated stromal cell-conditioned medium (CM) but was only capable of neutralizing 62%-91% of the bioactivity in endotoxin-stimulated, irradiated stromal cell-CM. Antisera against rGM-CSF and rIL3 demonstrated variable effects. When the antisera were used in combinations, only the mixture of anti-CSF-1 + anti-GM-CSF resulted in 100% neutralization of the activities in endotoxin-stimulated, irradiated stromal cell-CM. This CM stimulated the IL3/GM-CSF-responsive cell line FDC-P1 but not the IL3-responsive (GM-CSF-unresponsive) cell line 32D cl-23. The FDC-P1 growth-promoting activity was inhibited only by the antiserum against GM-CSF and not by antiserum against IL3. These experiments indicate that stromal cells from long-term bone marrow cultures can produce and release CSF-1 and GM-CSF while the production of IL3 in this system, if there is any, could not be demonstrated.