Recombinant human interleukin-11 directly promotes megakaryocytopoiesis in vitro

We have investigated the mechanism of action of the thrombopoietic cytokine, recombinant human interleukin-11 (rhIL-11), on megakaryocytopoiesis in vitro. We have shown that rhIL-11-induced murine and human megakaryocytopoiesis are not mediated by thrombopoietin (Tpo). Murine megakaryocytes (MKs) were produced from bone marrow (BM) mononuclear cells cultured with rhIL-11, IL-3, and a combination of the two cytokines. Conditioned media (CM) were collected and assayed for the presence of biologically active Tpo. Tpo activity was not detected in any of the CMs tested. Next, human BM CD34+ cells were cultured in serum-free fibrin clot medium with rhIL-11, IL-3, or rhIL-11 plus IL-3 and an antibody that neutralizes human Tpo activity. No inhibition of either burst-forming unit-MK- or colony-forming unit-MK-derived colony formation was observed. The antibody did partially inhibit steel factor-induced MK-colony formation, suggesting that the actions of this cytokine are mediated, in part, by Tpo. We determined that MKs can be direct targets of rhIL-11 by showing the expression of functional IL-11 receptor on these cells. Total RNA was prepared from cultured human BM CD41+CD14- cells (MKs) and IL-11 receptor alpha chain mRNA was detected in the MKs by reverse transcription-polymerase chain reaction. Analysis of single-sorted CD41+CD14- cells confirmed that the observed IL-11 receptor expression was not due to contaminating CD41- cells in the pool. The presence of rhIL-11 receptor alpha chain protein in the cells was established by Western blot analysis. After a short exposure of purified BM MKs to rhIL-11, enhanced phosphorylation of both its signal transduction subunit, gp130, and the transcription factor, STAT3 was detected, showing a direct activation of receptor signaling by the cytokine. Consistent with the lack of effect of rhIL-11 on platelets in vivo, IL-11 receptor alpha chain mRNA and protein were not detected in isolated human platelets. These data indicate that rhIL-11 acts directly on MKs and MK progenitors but not on platelets.