While CX3CR1 is clearly involved in their survival, S1PR5 is rather implicated in their egress from the BM although it may also contribute indirectly in their survival. Finally, we investigated the role of S1P in the physiology of Ly6C− monocytes using in vitro and in vivo experiments. In vitro, we measured responsiveness of monocytes to S1P gradients in chemotaxis chambers. No consistent migration of either population of monocytes was observed (Fig. 5A),

whereas both monocyte populations migrated in response to CCL2 gradients (Fig. 5B). In the same experiments, NK cells migrated in response to both S1P and CCL2 gradients (Fig. 5A and B), as JQ1 previously reported [16]. WT and S1pr5−/− Ly6C− monocytes migrated equally to CCL2 gradients, excluding a possible cross talk between CCR2 and S1PR5 (Fig. 5C). We also cultured Ly6C− monocytes with S1P at concentrations similar to those observed in vivo. The addition of S1P at any concentration did not change monocyte viability in vitro (Fig. 5D and data not shown). Next, we treated mice with the sphingosine lyase inhibitor deoxypyridoxine (DOP), which has been shown to dramatically increase S1P levels in tissues and disrupt

S1P gradients in vivo [22]. Upon treatment with DOP, peripheral T-cell numbers dropped, as previously reported [22]. However, DOP had no effect on the trafficking or the number of Ly6C− monocytes (Fig. 5E) and NK cells [22] even CT99021 after prolonged (10 days) treatment (Fig. 5E). The ex vivo viability of blood and BM Ly6C− monocytes was not modified either (Fig. 5F). Altogether, these results suggest that S1P and S1P gradients are not involved in monocyte Celastrol survival and unexpectedly not in their trafficking. In this article, we report for the first time a high expression of S1PR5 in patrolling monocytes and the paucity of these cells in the peripheral compartment of S1pr5−/− mice. The following body of evidences supports a role for S1PR5 in BM egress of patrolling monocytes: (i) We previously showed

that S1PR5 was involved in NK-cell egress from the BM to blood [20, 23]. Moreover, several other members of the family of S1P receptors (S1PR1, S1PR3) are clearly involved in egress of different leukocyte subsets from central and peripheral lymphoid organs [24]. (ii) Ly6C− monocytes are reduced in BM sinusoids of S1pr5−/− mice, whereas they are preserved, or even slightly increased in Cx3cr1gfp/gfp mice, which only exhibit impaired survival of Ly6C− monocytes at the periphery. (iii) The phenotype of S1pr5−/− mice is very similar to that of Ccr2−/− mice in which monocyte egress from the BM has been shown to be clearly impaired [15]. In particular, the number of Ly6C− monocytes was normal in the BM of S1pr5−/− and Ccr2−/− mice but reduced in the blood circulation and in BM sinusoids.