Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6

We have previously demonstrated that interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) stimulate various aspects of megakaryocytopoiesis. We have investigated the capacity of interleukin 6 (IL-6) to stimulate megakaryocyte colony formation from both normal Balb/C marrow and light-density marrow extensively depleted of adherent, pre-B, B and T cells. Human recombinant IL-6 (167 ng/ml) stimulated megakaryocyte colony formation from normal marrow (8.6 +/- 1 megakaryocyte colony-forming units [CFU-meg]/10(5) cells) as compared to control (1.5 +/- 4 CFU-meg/10(5) cells) in 16 determinations (p less than 0.01). IL-6 (167 ng/ml) also stimulated CFU-meg formation from depleted marrow (control, 10.8 +/- 4 CFU-meg/10(5) cells versus IL-6, 68 +/- 19 CFU-meg/10(5) cells in 12 determinations, p less than 0.01). IL-6 synergistically augmented IL-3-induced colony formation (139% IL-3 control, 120% calculated IL-3 plus IL-6 control, n = 11, p less than 0.01) in normal marrow and showed an additive effect in depleted marrow (133% IL-3 control, p less than 0.01, 114% of IL-3 plus IL-6, value not significant [NS] at 0.05 level). Studies with recombinant murine IL-6 gave similar results. There was an increasing level of megakaryocyte colony-stimulating activity from G-CSF (16,667 U/ml, 2.47 +/- 0.6 CFU-meg/10(5) cells, n = 17), to IL-6 (167 ng/ml, 8.47 +/- 0.96 CFU-meg/10(5) cells, n = 19), to GM-CSF (52 U/ml, 23 +/- 4 CFU-meg/10(5) cells, n = 14), to IL-3 (167 U/ml, 48 +/- 5 CFU-meg/10(5) cells, n = 20) as compared to media-stimulated marrow (range 1.29-1.86 CFU-meg/10(5) cells). A similar hierarchy was seen with depleted marrow. Combinations of factors (including IL-3, GM-CSF, G-CSF, and IL-6) tested against normal unseparated murine marrow did not further augment CFU-meg numbers over IL-3 plus IL-6 but did increase colony size. These data suggest that IL-6 is an important megakaryocyte regulator, that at least four growth factors interact synergistically or additively to regulate megakaryocytopoiesis, and that combinations of growth factors, possibly in physical association, might be critical in stimulating megakaryocyte stem cells.