Monocyte-derived CXCL7 peptides in the marrow microenvironment

Abstract

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoieticderived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The recombinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis. ( IntroductionHematopoietic regulation takes place within the marrow microenvironment (ME), which is defined by function as a complex of cells and cell products critical for the maintenance and regulation of stem cells and their progeny. The ME contains mesenchymal cells (eg, fibroblasts, adipocytes, and smooth muscle, all referred to as stromal cells); it also contains hematopoietic-derived monocytes/ macrophages. Primary long-term cultures (LTCs) of marrow stroma have been used to approximate the ME in vitro. 1 The model has proven useful for identifying cells and factors that play a role in supporting hematopoiesis. However, the heterogeneity of the cell types that comprise a functional LTC have led many investigators to generate stromal cell lines to dissect ME functional components. [2][3][4] We have previously reported on immortalized stromal lines generated by transducing human stromal cells with E6/E7 genes from human papilloma virus (HPV). 5,6 Two of these lines have been studied and characterized in detail. One, designated HS27a, supports maintenance of early hematopoietic progenitors in "cobblestone areas" and does not secrete growth factors. The other, designated HS5, does not support cobblestone areas but does secrete copious amounts of growth factors. These lines have hence been hypothesized to represent cell types that contribute to functionally distinct niches.Monocyte-derived macrophages have long been known as critical components of both the in vivo and in vitro ME. In vivo, the macrophage can function as the "nurse cell" of the ery...

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