Mice carrying the hypomorphic Gata1low mutation develop myelofibrosis with age. We reported that megakaryocytes (MK) from Gata1low mice, although expressing normal levels of P-selectin, abnormally retain the protein on the demarcation membrane system. Such defective localization triggers pathological emperipolesis of neutrophils within the MK, death of Mk by neutrophil protease-induced para-apoptosis, and release of TGF-β in the microenvironment (

Centurione et al, Blood 104:3573, 2004
). These data lead to the hypothesis that these mice develop fibrosis because of TGF-β-mediated fibroblast activation. This hypothesis is further analyzed here by determining the amount of TGF-β present in the microenvironment and by characterizing the state of fibroblast activation in spleens from Gata1low mice during disease progression. ELISA on organ washes revealed modest (2-fold) increases of TGF-β. In contrast, the number of TGF-β-gold particle in the spleen of these animals increased with age from ~20 (6-month), to ~230 (13-month) per field (5 and 50-times higher than normal, respectively). In the extracellular space, TGF-β-gold particles were specifically associated with the non-overlapping regions of the collagen polymers. It is possible that the detection of TGF-β was underestimated by ELISA because washing with a mild detergent solution does not remove the factor from the collagen fibers. With disease progression, the frequency of TGF-β particles in the cytoplasm of all the cells, including the fibroblasts, surrounding the collagen fibers increased as well. Furthermore, fibroblasts surrounding these “TGF-β-collagen spots” acquired an activated morphology that included high expression of fibronectin and collagen (by immunogold staining), and development of long protrusions (up to 3–4 per cell). Through these protrusions, fibroblasts established close contacts with MK in a process that could involve either close (short range) or distal (long range) cell peripolesis. In both cases, the protrusions penetrated within the MK releasing fibronectin and collagen in its cytoplasm. This previously undescribed process of fibroblast-MK peripolesis ultimately resulted in collagen accumulation in the MK cytoplasm, and death of the cell by para-apoptosis. Myeloperoxidase and TGF-β co-localization studies indicated that fibroblast peripolesis and neutrophil emperipolesis of Gata1low MK were independent and not mutually exclusive. However, when they involved the same MK, the peripoletic fibroblast contained myeloperoxidase-gold particles and died of para-apoptosis as well. These data indicate that the primary defect induced by the Gata1low mutation at the MK level triggers two pathological cell-MK interactions: the first one, neutrophil emperipolesis, directly by abnormal cell localization of an adhesion protein, and the second one, fibroblast peripolesis, indirectly, by increasing TGF-β in the microenvironment. We suggest that the altered growth factor milieu of the microenvironment, due to the presence of "collagen-growth factor spots", and pathological fibroblast-MK peripolesis both contribute to the patho-biology of myelofibrosis in Gata1low mice. (On behalf of the MyeloProliferative Disorders Research Consortium).

Topics:
adhesions, apoptosis, disease progression, endopeptidases, fibroblasts, fibronectins, fibrosis, gold, growth factor, megakaryocytes

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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