Macrophage-Associated Mesenchymal Stem Cells Figure 2. Macrophage-secreted soluble factors induce IL-6 and CXCL10 secretion by MSCs. Macrophages and MSCs have distinct cytokine secretion profiles that are variable and significantly dependent on the soluble factors and cells in their local environment. Both macrophages and MSCs are highly plastic and assume unique phenotypes when activated by paracrine factors. MSCs cultured in macrophage-conditioned medium for 24 hours assume a pro-inflammatory phenotype with increased secretion of IL-6 and CXCL10. Resting MSCs secrete 394 pg/mL and an undetectable amount of IL-6 and CXCL10, respectively. Upon activation by macrophage-conditioned medium, MSCs increased their secretion of IL-6 to 31,213 pg/mL and CXCL10 to 13,906 pg/mL. This represents a 56-fold increase in IL-6 (p,0.0001) and a 1.4-fold increase in CXCL10 (p,0.003). In addition, MSCs minimally increased the secretion of CCL5 by 1.2-fold from 287 pg/mL to 345 pg/mL (p ,0.02). doi:10.1371/journal.pone.0035036.g002 (Figure 5A), while 5 ng/mL CCL2 recombinant protein stimulated a 36% increase in MSC chemotaxis (Figure 5B). The addition of 10 ng/mL IL-8 increased MSC chemotaxis from 69.7+/210.4 to 118.5+/210.6 MSCs, a 1.7-fold increase (Figure 5C). While CCL5, CCL2, and IL-8 all induce MSC migration, the addition of any of these factors alone does not replicate the 5- to 9-fold increase in MSC chemotaxis seen in response to dU937 CM (604+/2 47.7 migrating MSCs). We then investigated whether CCL5, CCL2, or IL-8 were required for the induction of MSC migration. A neutralizing antibody directed against CCL-5 attenuated, but did not completely block the response of MSCs to macrophage CM. The addition of 10 mg/mL anti-CCL5 reduced MSC chemotaxis by 27% (Figure 5D), while 10 mg/mL anti-CCL2 did not significantly reduce chemotaxis (Figure 5E). A neutralizing antibody directed against IL-8 showed a trend toward reduced MSC chemotaxis from 604+/2147.7 MSCs to 374+/2125.9, however this was not statistically significant (Figure 5F). Activation of JNK is Required for the Stimulation of MSC Migration by Macrophages Because individual external stimuli could not maximally induce MSC migration, we investigated the role of second messenger pathways in this response. P38 MAPK, MAPK/ERK, and SAPK/JNK pathways are involved in chemotaxis in a number of systems [15–19]. We characterized the impact of inhibition of these signaling pathways on macrophage-induced MSC chemotaxis. Increased migration of MSCs was seen in response to macrophage CM compared to control medium (2,216+/2212, n = 3) and (471+/26, n = 3), respectively. Che- motaxis was unaffected by ERK or p38 inhibition (Figure 6A and 6B). However, JNK inhibition significantly reduced the migration of MSCs in response to dU937 CM to 987 (+/23.5, n = 3), a 55% decrease (Figure 6C and 6D). Trypan blue staining verified that the effect on cell migration was not due to changes in cell viability (Figure 6E). None of the inhibitors used significantly decreased MSC viability at 25 mM. PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35036