The liver contains mDCs and tissue macrophages that regulate local immune responses.

Under basal conditions, these cells are continually replenished by the recruitment of precursors from blood, and this increases with inflammation. We show that CD16+ cells redistribute in the chronically inflamed liver and can be detected at sites of inflammation and fibrosis (Fig. 3). This is consistent with studies suggesting that CD16+ cells are the precursors check details of inflammatory tissue macrophages and inflammatory DCs.13 Human CD16+ cells undergo differentiation into DCs after migration through endothelium,10, 16, 18 and CD16+ DCs are present in the human liver,41 leading us to investigate the molecules involved in CD16+ monocyte recruitment from blood

into the liver through hepatic sinusoids. We used flow-based adhesion assays incorporating primary human hepatic sinusoidal endothelium to model the liver sinusoid.27 We showed that human CD16+ monocytes express a cell adhesion molecules and chemokine receptors that could promote recruitment from blood, including CCR4, CXCR4, CX3CR1, CD18, and CD49d. We then demonstrated that they use a combination of CX3CL1, VCAM-1, PI3K cancer and VAP-1 to arrest on HSECs from flow and a combination of CX3CR1 and VAP-1 to undergo transendothelial migration. Thus, we show for the first time that VAP-1 is involved in DC precursor recruitment to peripheral tissue and define the role played by CX3CR1 in transmigration through human endothelium under flow. CX3CR1, which is expressed at uniformly high levels on CD16+ monocytes (Fig. 1), was the dominant chemokine receptor involved (Figs. 4 and 5) and antibodies against CX3CR1 inhibited both adhesion and transmigration across HSECs. CX3CL1 is a transmembrane chemokine that can support adhesion independently of signaling through its GPC receptor, consistent with our finding that adhesion of CD16+ cells on HSECs is more efficiently inhibited by anti-CX3CL1 antibodies than PTX. However, transmigration was clearly

dependent on signaling by way of CX3CR1, because it was almost completely prevented by PTX. To investigate the role of CX3CL1 further, we studied adhesion under flow to recombinant proteins. In this system, CX3CL1 was unable to support adhesion on its Racecadotril own but facilitated adhesion when coimmobilized with VCAM-1 by activating the α4β1-integrin. The recombinant CX3CL1 used in these experiments is not fully glycosylated, and because GPC-independent adhesion is mediated by the mucin domains that decorate transmembrane CX3CL1, this may explain its inability to directly support adhesion. CX3CL1 has been shown to support monocyte adhesion to human umbilical vein endothelial cells under flow,17 and our observations extend its role to a liver-specific vascular bed and implicate it in transendothelial migration.