Increased and Pathological Emperipolesis of Neutrophils within Megakaryocytes Associated with Myelofibrosis in GATA-1^Low_Mice.

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 (Gata1^tm2Sho or GATA-1^low 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-1^low mutation blocks Mk maturation between stage I and II, resulting in accumulation of defective Mk in the tissues of GATA-1^low 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-1^low 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-1^low Mk contain 1-3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1^low 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.