Conditioned medium from cultures of unstimulated mature osteoclasts contained a variety of chemokines, including MCP-1/CCL2, GROα/CXCL1 and IL-8/CXCL8 (Fig. 1A), indicating that osteoclasts had the capacity to recruit immune cells, including T cells and NK cells (via MCP-1/CCL2), and granulocytes (via GROα/CXCL1 and IL-8/CXCL8). Other factors produced by unstimulated osteoclasts detected on the array included IL-1RA, soluble ICAM-1 (sICAM-1) and Serpin E1. We also quantified production of a variety of chemokines and detected marked levels of MCP-1/CCL2 (753.02 ± 170.17 pg/ml), IL-8/CXCL8 (606.43 ± 44.95 pg/ml) and RANTES/CCL5

(331.81 ± 18.42 pg/ml) in osteoclast conditioned medium, thereby further Selleckchem Osimertinib supporting the idea that osteoclasts are capable of influencing the recruitment of a variety of immune cells. We then sought to determine if soluble mediators released by osteoclasts could induce the migration of γδ T cells. Due to the potential confounding effects of FBS present in conditioned medium for stimulating T cell migration directly, we generated conditioned medium from osteoclasts cultured for 48 h in SCH772984 molecular weight the absence of serum but supplemented with M-CSF and RANKL; conditions which did not adversely affect osteoclast viability as assessed by cellular morphology (data not shown). γδ T cells were pre-activated with 100 U/ml IL-2 for 12 h prior to addition, since unstimulated γδ T cells had

limited motility in response to FBS-induced migration (data not shown), consistent with a previous study of T cell chemotaxis [22]. While activated γδ T cells did not migrate

towards serum-free medium (Fig. 1B), FBS induced marked γδ T cell migration (~ 15–20% of input cells — data not shown). Interestingly, serum-free osteoclast conditioned medium also induced marked migration of γδ T cells across the Transwell membrane, comparable to that observed with FBS, indicating that osteoclasts release soluble factors capable of inducing the migration of γδ T cells. We next assessed whether osteoclasts could induce activation of T cells, using the early activation marker CD69. When γδ T cells or CD4+ T cells were co-cultured with Alectinib osteoclasts for 3 days a significant increase in CD69 expression was observed in both the γδ T cell (Fig. 2A) and CD4+ T cell populations (Fig. 2B). A non-significant trend for macrophages to induce CD69 expression on both γδ T cells (Fig. 2A) and CD4+ T cells (Fig. 2B) comparable to that observed with osteoclasts was also demonstrated. Following co-culture with treated osteoclasts (i.e. osteoclasts pre-treated with TNFα and IFNγ for 24 h), CD69 expression was further increased on γδ T cells, although this was not statistically different from untreated osteoclasts. A similar further upregulation of CD69 expression on CD4+ T cells was also observed following co-culture with treated osteoclasts.