Inhibition of CXCR1/2 reduces the emperipolesis between neutrophils and megakaryocytes in the Gata1 low model of myelofibrosis.

Emperipolesis between neutrophils and megakaryocytes was first identified by transmission electron microscopy. Although rare under steady state conditions, its frequency greatly increases in myelofibrosis, the most severe of the Myeloproliferative Neoplasms where it is believed to contribute to increasing the TGF-β microenvironmental bioavailability responsible for fibrosis. The challenge of performing studies by transmission electron microscopy has hampered up to now the study of factors which drive the pathological emperipolesis observed in myelofibrosis. We established a user-friendly confocal microscopy method that detects emperipolesis by staining with CD42b, specifically expressed on megakaryocytes, coupled with antibodies that recognize the neutrophils (Ly6b or neutrophil elastase antibody). With such an approach, we first confirmed that the bone marrow from myelofibrosis patients and from Gata1 low mice, a model of myelofibrosis, contain great numbers of neutrophils and megakaryocytes in emperipolesis. Both in patients, and in Gata1 low mice, the emperipolesed megakaryocytes were surrounded by high numbers of neutrophils, suggesting that neutrophil chemotaxis precedes the actual emperipolesis event. Since neutrophil chemotaxis is driven by CXCL1, the murine equivalent of human interleukin-8 that is expressed at high levels by malignant megakaryocytes, we tested the hypothesis that neutrophil/megakaryocytes emperipolesis could be reduced by Reparixin, an inhibitor of CXCR1/CXCR2. Indeed, the treatment greatly reduced both neutrophils chemotaxis and their emperipolesis with the megakaryocytes in treated mice. Since treatment with Reparixin was previously reported to reduce both TGF-β content and marrow fibrosis, these results identify neutrophil/megakaryocyte emperipolesis as the cellular interaction that links interleukin-8 to TGF-β abnormalities in the pathobiology of marrow fibrosis.