Abstract
CXCL7 is a 14.0 Kd chemokine produced by megakaryocytes. There are 4 different peptides derived from the pro-peptide by cleavage of the amino terminal, each with distinctive functions: The longest form, LDGF (Leukocyte Derived Growth Factor) is mitogenic for fibroblasts while the smallest, Neutrophil Activating Peptide-2 (NAP-2) inhibits megakaryocytopoiesis. Therefore both forms have, in theory, the potential to influence the composition of the marrow microenvironment. We report here that an additional source of CXCL7 is the monocyte, which upregulates CXCL7 mRNA several fold when stimulated by stromal cells. Briefly, flow sorted CD 14+ monocytes were cultured alone and in co-culture with stromal cell lines HS-5 and HS-27a. Between group comparisons of gene expression profiles were made to identify genes up or down regulated in co-cultures. Expression of CXCL7 was found to be upregulated 25 fold when monocytes were co-cultured with HS-5 and 13 fold with HS-27a as compared to monocytes cultured alone. The array data was confirmed by RT-PCR. Using transwell cultures it was possible to demonstrate that the monocytes and not the stromal cells were responsible for CXCL7 gene expression, which peaked at 6 days. ELISA-based assays detected CXCL7 peptides in the supernatants of monocyte/stroma co-cultures after day 2 of culture. Levels detected ranged from 10 to 50 ng/ml, which are physiologically relevant concentrations. Western Blot analysis showed the 3 day cultures had predominantly LDGF; by day 6 the smaller form, NAP-2 was also present. Since peptide cleavage involves Cathepsin and related proteinases, this delayed appearance of NAP-2 may suggest that the required concentrations of proteinases accumulate with time. The presence of both mRNA and secreted protein in primary long-term cultures of human bone marrow, (peptide levels peaking at 125 ng/ml on day 22) suggest that normal, non-immortalized stroma also induces expression of the CXCL7 gene in the monocytes. Stroma-derived molecules known to stimulate monocytes (IL-1beta, IL-6, GM-CSF and G-CSF) were evaluated for their ability to induce CXCL7 gene expression. Of those tested, only GM-CSF was shown to induce the expression of CXCL7, but at levels far lower than induced by HS-5. As reported previously addition of NAP-2 to CFU-Meg cultures decreased the CFU-Megs by up to 40% despite maximal TPO and the CFU-Megs that did grow were smaller than those in control cultures. Also, addition of flow-sorted monocytes to cultures of primary marrow stroma, not only resulted in CXCL7 gene expression, but significantly increased fibroblast growth in these cultures. In summary our data show that a stroma-derived signal induces the expression of CXCL7 gene products in monocytes. Given that the monocyte is a critical component of the marrow microenvironment we postulate that induction of CXCL7 by stromal signals can affect both structure and function of the marrow microenvironment by increasing fibrosis and decreasing platelet production. Preliminary data from monocytes of 5 patients with Myelodysplastic Syndrome (MDS) suggests that MDS derived monocytes have abnormal patterns of CXCL7 expression. Whether this contributes to the pathophysiology of MDS remains to be determined.
Integration of Genome and Chromatin Structure with Gene Expression Profiles To Predict c-MYC Recognition Site Binding and Function