Macrophage-Associated Mesenchymal Stem Cells Figure 5. Stimulation of mesenchymal stem cells by CCL5, CCL2, and IL-8 secretion induces migration. To determine the role of macrophage-secreted CCL5, CCL2 (MCP-1), and IL-8 in MSC migration, the level of migration in response to recombinant proteins was analyzed. CCL5 (A), CCL2 (B), and IL-8 (C) significantly increased the number of migratory MSCs in a standard Boyden chamber chemotaxis assay. To further characterize the role of CCL5, CCL2, and IL-8 in macrophage-induced MSC migration, neutralizing antibodies targeting these factors were added to macrophage CM prior to migration. While each individual factor induced MSC migration, only CCL5 inhibition reduced MSC migration in response to macrophage CM (D, E, F). doi:10.1371/journal.pone.0035036.g005 and chemokines induce MSC migration [39] and signals for MSC migration are likely to vary depending on local conditions. Recent evidence demonstrates that Akt, ERK, and p38 signaling pathways may be involved in CXCL12-mediated MSC migration. The ERK and p38 signaling pathways have been implicated in the mobilization of MSCs in response to an array of factors including CXCL12 and hepatocyte growth factor[40–43]. The JNK signaling pathway has been shown to be involved in the migration of astrocytes [44], tumor cells [45], smooth muscle cells [46,47], neutrophils [48] and recently in MSC chemotaxis [49,50]. Our work demonstrates that macrophages induce MSC migration through the production of a constellation of soluble factors. Individually, CCL2, CCL5, and IL-8 stimulate MSC migration. This system has significant redundancy, as no single factor tested could induce maximal MSC migration. However, a common molecular thread for the interaction between macrophages and MSCs appears to be activation of JNK. Small molecule inhibitors targeting the ERK, p38, and JNK transcription factors were used to determine specific pathways involved in MSC chemotaxis. While the individual blockade of several cytokine receptors failed to significantly impair MSC migration, inhibition of JNK activation drastically reduced the response of MSCs to macro- phages. This is distinct from other studies of MSC migration, which have focused on signaling through the ERK, p38 and Akt pathways [51]. Based on these results we suggest that targeting JNK in MSCs may represent a unique avenue to manipulating their response to the inflammatory microenvironment. Studies to characterize JNK-induced MSC chemotaxis to other stimuli and the investigation of JNK-inhibited MSC localization to injury models in vivo are currently underway. Our data suggest that macrophages induce a migratory MSC phenotype with increased secretion of CXCL10, CCL5, and IL-6. While this work provides a framework for understanding the impact of macrophages on MSC phenotype, other classes of macrophages may have differential effects on MSC biology. Further exploration of the interaction between macrophages and MSCs in different microenvironments will lead to a more complete understanding of the mechanisms of MSC response during processes such as tissue repair and tumor growth and will provide the framework for targeted gene and drug delivery, tissue regeneration, and novel targets for promotion or inhibition of MSC chemotaxis. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35036