Macrophage-Associated Mesenchymal Stem Cells Figure 1. Inflammatory cytokine gene expression by MSCs is affected by macrophage-secreted soluble factors. MSCs exposed to macrophage CM for 24 hours express an altered inflammatory cytokine gene expression profile. Analysis of 84 key genes mediating the inflammatory response was conducted using RT-PCR generating a heat map expressing log-fold changes. Gray boxes represent genes that were undetected in either the control MSCs or the macrophage CM-stimulated MSCs (A). When stimulated by macrophage CM, MSCs increase the expression of inflammatory factors such as, CCL7, IL-8, CCL20, CXCL6, CCL2, CCL5, CXCL1, and CXCL3. The changes ranged from 2.1 to 12.9-fold (B). These data can also be visualized in a scatter plot, showing gene up-regulation as points scattered into the upper left quadrant (C). doi:10.1371/journal.pone.0035036.g001 produce macrophage-activated MSCs, cell-free macrophage conditioned medium (CM) was harvested and added to MSC cultures for 24 hours. IL-6 levels in the medium increased from 394.3 pg/mL to 31,213.3 pg/mL and levels of CXCL10 and CCL5 were increased by 1.4- and 1.2-fold, respectively. Despite an increase in mRNA expression, IL-8 and CXCL12 did not show significant changes in secretion and CCL7 secretion was decreased despite a marked elevation in mRNA levels (Figure 2). Macrophages Secrete Soluble Factors that Stimulate MSC Migration We then investigated the ability of soluble factors produced by macrophages to induce MSC migration in vitro. Following an 18- hour migration, conditioned medium from both the human lymphoma cell line U937 differentiated to a macrophage phenotype (dU937) and primary culture human macrophages stimulated MSC migration (5.7- and 5.3-fold compared to control medium) (Figure 3A). The ability of dU937 macrophages to induce MSC migration was compared with undifferentiated U937 cells (Figure 3B). Conditioned medium from U937 cells cultured without PMA (undifferentiated U937) stimulated a 2.9-fold increase in MSC migration compared to control medium. dU937 CM promoted a significantly higher level of MSC migration, inducing an 8.7-fold increase over control medium-stimulated MSCs. Macrophages Secrete an Array of Factors with the Capacity to Induce Migration of MSCs, Including CCL5, CCL2, and IL-8 In order to identify factors involved in stimulating MSC chemotaxis, the cytokine profile of macrophage CM was determined. Macrophages secrete IL-8 (1,162 pg/mL), CCL2 (150 pg/mL), CCL5 (71 pg/mL), VEGF (260 pg/mL), and CXCL12 (643 pg/mL)(Figure 4A). Differentiated U937 cells displayed a cytokine profile with a similar pattern consisting of IL-8 (18,630 pg/mL), CCL2 (24,246 pg/mL), CCL3 (206 pg/ mL), CCL5 (1,156 pg/mL), VEGF (6,011 pg/mL), and CXCL12 (3,970 pg/mL)(Figure 4B). CCL5, CCL2, and IL-8 are Chemotactic for MSCs CCL5, CCL2, and IL-8 were present in increased levels in both primary macrophage CM and dU937 CM when compared with control medium. The ability of CCL5, CCL2, and IL-8 to act as a stimulus for MSC migration was tested. The addition of 10 ng/mL recombinant CCL5 induced a 39% increase in MSC chemotaxis compared to control medium PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35036