Tie2 Expressing Monocytes in the Spleen of Patients with Primary Myelofibrosis

Abstract

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

Fig 1. Cytofluorimetric analysis of spleen tissue derived TEMs.

CD14^lowCD16^bright non classical monocytes from a patient with PMF were gated (panel A); CD14^lowCD16^bright not expressing the CD62L and CCR2 antigens on the membrane (panel B) were than evaluated for the expression of Tie2 (panel C). Panels D, E, F for one representative healthy subject (CTRL).

Fig 2. Confocal microscopy images of spleen tissue samples stained with anti-CD14 and anti-Tie2.

Spleen tissue samples were immunostained for the indicated markers and a small capillary from each stained specimen was chosen for display. A) Arrow heads indicate cells expressing both CD14 in red (i) and Tie2 in green (ii) in the perivascular area of spleen tissue sample from 1 representative patient with primary myelofibrosis (PMF). The yellow colour indicates colocalization of CD14 and Tie2 (iii). B) Thin arrows indicate CD14⁺ cells (iv) not expressing Tie2 (v, vi) in spleen tissue sample from 1 representative CTRL. DAPI (blue) was used as the nuclear marker and the images were incorporated in the merge panels (iii, vi). Images were obtained at 63x magnification.

Fig 3. In vitro tubulogenesis induced by spleen MNC or selected CD14⁺ cell CM.

Digital images of endothelial tubes obtained by bright-field light microscopy 24 hours after plating Endothelial Colony Forming Cells from a healthy donor on Matrigel-coated wells in presence of CM of spleen tissue derived MNCs from two patients with primary myelofibrosis (PMF) and one representative CTRL (panel A), or CM of spleen tissue derived selected CD14⁺ cells from one representative patient with PMF and one CTRL (panel B). Cultures were examined under an inverted microscope (Labovert, Leitz, Germany) in bright field, at 2.5X magnification using a PL (Leitz Wetzlar, Germany) objective; quantitative evaluation of the tube-like structures, expressed as ratio of the total length of tubular structures per field (mm)/surface of the field (mm²) (ImageJ software by National Institutes of Health, USA, http://rsbweb.nih.gov/ij/.), of all the patients with PMF and CTRLs tested is shown (panel C).