Thrombopoietin Regulates Proliferation and Maturation of Murine Mast Cells.

Abstract

Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med 197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.