SIMULTANEOUS ACTIVATION OF SIGNALS THROUGH GP130, C-KIT, AND INTERLEUKIN-3 RECEPTOR PROMOTES A TRILINEAGE BLOOD-CELL PRODUCTION IN THE ABSENCE OF TERMINALLY ACTING LINEAGE-SPECIFIC FACTORS

We assessed the biologic role of signaling through gp130, a signal-tra nsducing receptor (R) component, in human hematopoiesis in vitro. Alth ough peripheral blood-derived CD34(+) cells ubiquitously expressed gp1 30 and interleukin-3 receptor alpha (IL-3R alpha), IL-6R alpha was onl y detected on 80% of these CD34(+) cells. We sorted CD34(+)IL-6R(+) or CD34(+)IL-6R(-) cells and studied the effect on hematopoietic colony formation of signaling through gp130 activated by IL-6 or a combinatio n of IL-6 and recombinant soluble human IL-6R (sIL-6R) in the presence or absence of stem cell factor (SCF) and/or IL-3. Signals activated b y SCF, IL-6, or IL-6/sIL-6R complex alone did not induce significant c olony formation. However, a combination of IL-3, SCF, and IL-6/sIL-6R complex dramatically induced many neutrophil (colony-forming unit-gran ulocyte [CFU-G]), erythroid burst (burst-forming unit-erythrocyte [BFU -E]), erythrocyte-containing mixed (CFU-Mix), and megakaryocyte (CFU-M eg) colony formations when CD34(+)IL-6R(-) cells were used as the targ et. CFU-G colony formation induced by the three signals was more evide nt when CD34(+)IL-6R(+) cells were used as the target. This distinct s ynergistic effect of the three different signals was confirmed by sing le-cell clone-sorting experiments. Moreover, colony formation (includi ng CFU-G, BFU-E, CFU-Mix, and CFU-Meg) was observed even in the presen ce of neutralizing antibodies for granulocyte colony-stimulating facto r, erythropoietin, and thrombopoietin (c-Mpl), whereas neutralizing an tibodies for gp130, IL-6R, IL-3, and SCF partially or completely block ed the synergistic effect. The maturation of neutrophilic, erythroid, and megakaryocytic cells supported by the three signals in serum-free cultures was confirmed by immunostaining using anti-CD66b, antiglycoph orin A, antihemoglobin alpha, and anti-CD41 monoclonal antibodies, res pectively. In contrast, any two of the three signals were insufficient for effective blood cell production in the absence of maturation fact ors. These results suggest that simultaneous activation of the three s ignals through gp130, c-kit, and IL-3R can induce in vitro proliferati on and differentiation of trilineage hematopoietic progenitors in the absence of terminally acting lineage-specific factors. (C) 1997 by The American Society of Hematology.