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Peripheral blood monocytes are a heterogeneous population of circulating leukocytes. Using a murine adoptive transfer system to probe monocyte homing and differentiation in vivo, we identified two functional subsets among murine blood monocytes: a short-lived CX₃CR1^loCCR2⁺Gr1⁺ subset that is actively recruited to inflamed tissues and a CX₃CR1^hiCCR2⁻Gr1⁻ subset characterized by CX₃CR1-dependent recruitment to noninflamed tissues. Both subsets have the potential to differentiate into dendritic cells in vivo. The level of CX₃CR1 expression also defines the two major human monocyte subsets, the CD14⁺CD16⁻ and CD14^loCD16⁺ monocytes, which share phenotype and homing potential with the mouse subsets. These findings raise the potential for novel therapeutic strategies in inflammatory diseases.