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The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.

blood

Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes