Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium

Abstract

Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony-stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti-GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF. In contrast, the Meg-CSA in aplastic human sera was completely resistant to neutralization by a 1/500 dilution of anti-IL-3. Anti-GM-CSF alone had a variable effect. When tested against two aplastic sera exhibiting high levels of Meg-CSA, a 1/500 dilution of anti-GM-CSF showed no inhibitory effect. However, the activity of four aplastic sera with relatively low levels of Meg-CSA was reduced by an average of 45% by the GM-CSF antisera. Addition of anti-IL-3 to the anti-GM-CSF did not further attenuate colony growth in these latter four aplastic sera. The use of higher concentrations of the GM-CSF and IL-3 anti-sera (dilutions of up to 1/20) also exhibited no additional inhibitory effects. Thus, all six aplastic sera demonstrated substantial Meg-CSA that was resistant to neutralization by anti-IL-3 and anti-GM-CSF. Other defined hematopoietic growth factors were evaluated for Meg-CSA in our culture system in order to determine if any one of them could account for the Meg-CSA in aplastic serum. Recombinant G-CSF, erythropoietin, IL-1α, IL-1β, IL-4, and IL-6 did not support megakaryocyte colony growth to any substantial degree. Thus, our data indicate that the Meg-CSA exhibited by PHA-LCM is composed entirely of IL-3 and GM-CSF. In contrast, a Meg-CSA is present in aplastic human sera that appears to be distinct from both IL-3 and GM-CSF, as well as from other defined hematopoietic cytokines. © 1990 by The American Society of Hematology.