Macrophage-Associated Mesenchymal Stem Cells Figure 4. Identification of soluble factors produced by macrophages. Unstimulated macrophages generated from human peripheral blood monocytes secrete IL-8, CCL2, and CCL5 (A) and do not secrete detectable amounts of IL-6 or MIP-1a. dU937 macrophages secrete increased levels of IL-8, CCL2, CCL20, CCL5, VEGF, and CXCL12 when compared with both control medium and undifferentiated U937 CM (B). All samples were analyzed in triplicate. *p,0.05. doi:10.1371/journal.pone.0035036.g004 polarization of MSCs to a motile phenotype with increased secretion of IL-6 and IP-10. Previous work has demonstrated functional interaction between MSCs and macrophages. Macrophages were initially broadly divided into M1 (classically activated macrophages) and M2 (alternatively activated macrophages) subtypes with subsequent expansion into a wide range of different phenotypes that are dictated by the local environment [35]. Co-culture of human monocyte-derived macrophages with MSCs led to increased cell surface CD206 expression and changes in cytokine production by the macrophages in response to stimuli including LPS, interferon gamma, and ionomycin and PMA. Mesenchymal stem cell educated macrophages had increased levels of IL-10 and IL-6 production and decreased levels of IL-12 and TNF-alpha in response to stimuli [36]. This constellation of markers suggests that MSCs induce a novel macrophage phenotype with characteristics of alternatively activated macrophages. In a murine model, bone marrow-derived MSCs suppressed the production of TNF-alpha, IL-6, and interferon gamma and increased production of IL-10 by LPS-stimulated peritoneal macrophages [37], again corresponding to a subtype of macrophages within the M2 spectrum [35]. It is possible that these differences in the response of macrophages to MSCs under various experimental conditions mimic the spectrum of macrophage response in diverse microenvironments in vivo. Thus there is accumulating data demonstrating changes in macrophage phenotype in response to MSCs. We present work that focuses on changes in MSC phenotype in response to macrophages. Two distinct MSC populations, MSC1 and MSC2, have been described and are induced by activation of toll-like receptor (TLR)-4 and TLR-3, respectively [38]. The priming of human MSCs with the TLR-4 agonist lipopolysaccharide (LPS) leads to a pro-inflammatory phenotype with increased secretion of IL-6 and IL-8 that was not able to suppress T-cell activation in culture. Stimulation of TLR-3 with poly(I:C; double-stranded RNA) led to elevated secretion of CCL5 and CXCL10 (interferon-gamma inducible protein-10) by MSCs with less significant increases in IL- 6 and IL-8 production. MSCs stimulated with poly(I:C; double stranded RNA) also inhibited T-cell activation in culture. MSC1 and MSC2 polarizations are likely two cellular profiles among an array of in vivo possibilities. Macrophage-associated MSCs assume a unique phenotype with some characteristics that more closely resemble MSC2 cells with increased CXCL10 and CCL5 secretion. However IL-6 secretion was also elevated 56-fold over control levels. Messenger RNA levels were increased for CCL7, IL-8, and CCL2 in macrophage-associated MSCs, however they were not secreted at increased levels. In addition to changes in the expression and secretion of soluble signaling molecules, MSCs also exhibited increased mobility in response to macrophage-derived factors. A number of cytokines PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35036