Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1low mice

Abstract

Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1 tm2Sho or GATA-1 low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1 low mutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1 low 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-1 low platelets. IntroductionMegakaryocytes (MKs) are specialized cells of the blood responsible for platelet production. 1 They originate from committed progenitor cells, usually localized in the marrow, through a complex maturation process, during which MK precursors progressively increase in size, while undergoing extensive synchronous morphologic changes in the cytoplasm and nucleus. 2 At the ultrastructural level, the major cytoplasmic modifications are represented by massive compartmentalization into discrete regions, delimited by intrusions of the plasma membranes, bound to give rise to the demarcation membrane system (DMS). 2 The DMS will, in turn, internalize platelet-specific ␣ granules, giving rise to proplatelets through a process defined thrombocytopoiesis. 3 In the meantime, chromosomes undergo several cycles of endo-duplications. As a result, the nucleus itself appears multilobed.Despite few differences, the morphologic changes associated with the maturation of MK precursors are similar between mice and humans. 4 On the basis of distinct ultrastructural characteristics, murine and human MK precursors are divided into 4 classes 2 : the promegakaryoblast, a small mononuclear cell expressing already platelet-specific proteins, such as von Willebrand Factor (VWF); the megakaryoblast (or stage I MK), a cell 15 to 50 m in diameter with a large, oval or kidney-shaped nucleus and several nucleoli, whose cytoplasm presents abundant ribosomes and a welldeveloped rough endoplasmic reticulum (RER); the promegakaryocyte (stage II MK), a cell 20 to 80 m in diameter with an irregularly shaped nucleus and a more abundant cytoplasm, containing a rudimental DMS; and, finally, mature megakaryocytes (stage III MKs) that contain a multilobed nucleus surrounded by abundant cytoplasm divided into a perinuclear (hosting the centrioles, few biosynthetically active organelles, and many ␣ granules), the intermediate (containing a well-developed DMS and platelet territories), and the peripheral (devoid of organelles and enriched of cytoskeletal proteins and microtubules) zone. 2 The complex process of MK maturation is controlled by lineagespecific extrinsic and intrinsic factors represented, respectively, by growth factors (such as thrombopoietin 5 [TPO]) and transcriptio...

Altered SDF-1/CXCR4 axis in patients with primary myelofibrosis and in the Gata1low mouse model of the disease

“…Marrow sections both from Gata1 low mice ( Figure 3A and 7,10 ) and from PMF patients 8 are characterized by an increased frequency of megakaryocytes which react strongly with the SDF-1 antibody (Figure 3 and 7). In the marrow of Gata1 low mice, the immunostaining was mainly localized within the megakaryocytes ( Figure 3A).…”

“…Megakaryocytes from Gata1 low mice and from PMF patients express, however, the same intrinsic abnormalities (i.e. higher frequency and higher retention in the marrow because of maturation retardation [6][7][8]56 and increased localization near the endosteum, in close spatial relationship with the osteoblasts 57 . All these factors can contribute to the increased megakaryocyte up-take of SDF-1.…”

“…Mice harboring the hypomorphic Gata1 low mutation, that impairs expression of this gene in megakaryocytes 4 , are born thrombocytopenic 5,6 . The megakaryocytes in the marrow from these mutants present the same morphological abnormalities as those observed in the marrow of PMF patients [7][8][9] . The mutants slowly develop with age all the hallmarks of the disease, including increased stem/progenitor cell trafficking and extramedullary hematopoiesis in liver 10,11 .…”

Abnormalities of GATA-1 in Megakaryocytes from Patients with Idiopathic Myelofibrosis

“…30,31 These include abnormal P-selectin localization on the demarcation membrane system, increased neutrophil emperipolesis with the subsequent release in the microenvironment of neutrophil proteases as well as of transforming growth factor-␤1 produced by megakaryocytes, and death by para-apoptosis. 32 Old (15 months on) GATA-1 low mice show features resembling human IM, that include: severe marrow fibrosis, osteosclerosis, thrombocytopenia and tear-drop poikilocytes on blood films, an increased number of circulating hematopoietic progenitors, splenomegaly, and extramedullary hematopoiesis. 24 Although alterations of the TPO receptor or of TPO regulatory pathways have not been documented in a well-characterized series of IM patients, 33 it has not been addressed thoroughly whether GATA-1 is mutated or abnormally expressed in IM.…”

Recombinant interferon-α in myelofibrosis reduces bone marrow fibrosis, improves its morphology and is associated with clinical response

“…Impaired emperipolesis of neutrophils may alter the intramegakaryocytic trafficking of α-granules, thus leading to the release of pro-fibrogenic growth factors (i.e., TGFβ and PDGF). 25,26 Our study shows that the administration of recombinant interferon-α can reduce both megakaryopoiesis (decrease megakaryocyte density) and granulopoiesis (decrease of cellularity, which in myelofibrosis is mainly related to the myeloid lineage). These observations may provide an explanation for the interferon-associated decrease of marrow fibrosis.…”