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Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1lowmutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize granules because von Willebrand factor is barely detectable in mutant MKs, and P-selectin, although normally expressed, is found frequently associated with the demarcation membrane system (DMS) instead of within granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in GATA-1low platelets. Mutant MKs are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process to establish contact with MKs by fusing their membrane with those of the DMS. As a result, 16% (in spleen) to 34% (in marrow) of GATA-1low MKs contain 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1low MKs have morphologic features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathologic neutrophil emperipolesis may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenvironment. (Blood. 2004;104:3573-3580)

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 Med197:281, 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.

GATA-1 is a transcription factor involved in the intrinsic control of erythroid, megakaryocytic and mast cell differentiation. Experimentally-induced deletion of the megakaryocytic (Mk)-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. The relationship, if any, between defective megakaryocytopoiesis and development of the disease is still poorly understood. Electron microscopy studies, coupled with immuno-electron microscopy, have allowed us to determine that the GATA-1low mutation blocks Mk maturation between stage I and II, resulting in accumulation of defective Mk in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize α-granules, since von Willebrand factor is barely detectable in mutant Mk while P-selectin, although expressed at normal level, is found mainly associated with the Demarcation Membrane System (DMS) rather than with granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in the megathrombocytes in the blood from GATA-1low mice. Both in marrow and spleen, mutant Mk are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process of establishing contact with the Mk by fusing their membrane with those of the DMS. As a result, 16 (in spleen) - 34 (in marrow) percent of GATA-1low Mk contain 1-3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1low Mk have morphological features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathological neutrophil emperipolesis represents one of the mechanisms leading to myelofibrosis by releasing Mk cytokines (TGF-β) and neutrophil proteases (myeloperoxidase) in the microenvironment which result, respectively, in marrow fibrosis and abnormal stem/progenitor cell trafficking.