Neutophil Gelatinase –Associated Lipocalin (LCN2) Affects Primary Melofibrosis Hematopoietic Progenitor Cells As Well As Microenvironmental Cells

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34⁺ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P<0.001) Media conditioned by PMF MNC contained higher levels of LCN2 than media conditioned by normal MNC (p=0.03). The LCN2 receptor level was flow cytometrically analyzed and a significantly greater percentage of normal MNC and CD34⁺ cells than PMF MNC and CD34⁺ cells expressed the LCN2 receptor. The effect of increasing concentrations of recombinant LCN2 on CFU-GM and BFU-E derived colony by normal BM CD34⁺ cells as well as the PMF CD34⁺ cells was evaluated. The addition of LCN2 inhibited CFU-GM and BFU-E derived colony formation by normal CD34⁺ cells at a dose of 100 ng/ml (p=0.048 and p=0.043, respectively), while a similar dose of LCN2 did not influence the number of colonies cloned from PMF CD34⁺ cells. Previously our laboratory has reported that the expression of the chemokine receptor CXCR4 was decreased in PMF CD34⁺ cells which we hypothesized contributed to abnormal trafficking of CD34⁺ cells. LCN2 has been reported to affect CXCR4 expression by marrow CD34⁺ cells (Costa D, et al. 2010, Cytokine). CXCR4 expression by PMF CD34⁺ cells was lower than that of normal BM CD34⁺ cells. Incubation with LCN2, however, further reduced the expression of CXCR4 of PMF CD34⁺ cells by 10 to 50 % (p=0.012). By contrast, LCN2 increased CXCR4⁺ expression by normal CD34⁺ cells. We next evaluated the effects of LCN2 on the BM microenvironment. Normal BM mononuclear cells were plated in dishes exposed to the vary doses of LCN2 (10, 50, 100, 200 ng/ml) and the formation of adherent cell layers was monitored. Low doses of LCN2 (10 and 50 ng/ml) promoted the formation and proliferation of adherent cell layers composed of fibroblast-like cells after 1-3 weeks of culture. The fibroblast-like cells expressed vimentin and von Willebrand factor, indicating that they resembled mesenchymal stem cells, fibroblast and endothelial cells. We then examined the formation of adherent cell layers by normal BM MNC co-cultured with PMF, PV or normal MNCs separated by a 0.4 um trans-well for three weeks. The proliferation of confluent fibroblast-like cells was observed solely in BM MNC co-cultured with PMF MNC. These data indicate that LCN2 is generated in increased amounts by PMF mononuclear cells and likely plays a role in PMF biology by promoting malignant hematopoiesis but suppressing normal hemaopoiesis, suppressing CXCR4 expression by PMF CD34⁺ cells and promoting marrow fibroblast proliferation.