Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells

Abstract

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14 ؉ peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up-or downregulated in the cocultures, with osteopontin (OPN) up-regulated more than 15-fold. The microarray OPN data were confirmed by Northern blot, real-time polymerase chain reaction (PCR), and by detection of OPN protein. High levels of OPN gene expression were also detected in 2-to 3-week-old primary LTCs. Using Transwells we determined that stromal cells were secreting a factor that upregulated OPN gene expression in CD14 ؉ cells. When CD34 ؉ cells were cultured in the presence of purified OPN, tyrosine phosphorylation of a 34-kDa molecule was increased 2-to 3-fold, an effect that was diminished in the presence of an OPN neutralizing monoclonal antibody. In addition, Notch1 gene expression was decreased 5-fold in OPN-treated CD34 ؉ cells. We conclude that interactions between stroma and monocytes can result in activities that limit the role of Notch signaling in hematopoietic regulation. IntroductionThe hematopoietic microenvironment (ME), defined largely by function, is a complex mixture of cells and factors critical for the regulation of hematopoiesis. The ME is assayed in vitro using primary long-term marrow cultures (LTCs) that consist of nonhematopoietic-derived stromal components as well as hematopoietic components, including monocytes and macrophages. A critical in vivo role for the macrophage in the marrow ME has long been inferred from histologic studies, best exemplified by the nurse cell of the erythropoietic island. In primary LTCs, up to 40% of all cells can be macrophages even after 12 weeks of culture. 1 These cells are known to secrete many cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF), all of which have direct effects on hematopoietic cells. Monocytes/macrophages are also capable of stimulating stromal cells to produce cytokines. [2][3][4][5] Because macrophages also produce transforming growth factor-␤ (TGF␤) and platelet-derived growth factor (PDGF), both of which can influence the mitotic rate of fibroblasts, the macrophage may also be capable of regulating stromal cell growth. [6][7][8][9][10] Gaining a more precise understanding of monocyte-stroma interactions within the ME is complicated by the heterogeneous nature of the stromal compartment. In an effort to functionally dissect the ME, several groups have established cloned stromal cell lines that identify individual ME components. [11][12][13][14][15] We previously reported on immortalized human marrow stromal ce...

Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells

“…[47][48][49] Our data demonstrate that a direct interaction between HSCs and Opn produced by osteoblasts results in ␤ 1 integrinmediated inhibition of cell proliferation. The recent studies of Iwata et al 50 suggest that the coculture of CD34 ϩ cells in the presence of the HS-27a stromal cell line plus monocytes in medium containing Opn results in a down-regulation of Notch1 after 4 days, which was inversely correlated to the amount of Opn present. Whether this effect is mediated by a direct interaction between Opn and CD34 ϩ cells or indirectly via the stromal cell line and or monocytes remains unclear.…”

Reduced expression of inducible gelatinase B/matrix metalloproteinase-9 in monocytes from patients with myelodysplastic syndrome: correlation of inducible levels with the percentage of cytogenetically marked cells and with marrow cellularity

“…[33][34][35][36][37] In a previous study, we investigated the interactions between monocytes and stroma in an in vitro coculture system using cloned human marrow stromal cell lines and normal peripheral-blood monocytes. 28,38 Transcriptome analysis identified MMP-9 as one of several genes strongly up-regulated in cocultures of HS-5 and CD14 ؉ cells (M.I. and B.T.-S., unpublished data, 2004).…”

“…CD14 ϩ cells were isolated by AutoMacs (Miltenyi Biotec, Auburn, CA) using a CD14 monoclonal antibody (clone TÜ K4; Dako, Carpinteria, CA) and anti-mouse immunoglobulin G2aϩb-conjugated magnetic microbeads (Miltenyi Biotec) as described before. 28 The purity of isolated CD14 ϩ cells from healthy donors and MDS patients was 98% (range, 94%-99%) and 82% (range, 60%-94%), respectively.…”

Osteopontin: a bridge between bone and blood

“…Independent in vitro studies have shown that Opn can directly and specifically affect haemopoiesis Stier et al, 2005 (Iwata et al, 2004). Hence, the microenvironment might downregulate Jagged1-Notch1 signalling which could then alter CD34 + cell-stromal interactions.…”