CX(3)CR1 and vascular adhesion protein-1-dependent recruitment of CD16(+) monocytes across human liver sinusoidal endothelium

Abstract

The liver contains macrophages and myeloid dendritic cells (mDCs) that are critical for the regulation of hepatic inflammation. Most hepatic macrophages and mDCs are derived from monocytes recruited from the blood through poorly understood interactions with hepatic sinusoidal endothelial cells (HSECs). Human CD16(+) monocytes are thought to contain the precursor populations for tissue macrophages and mDCs. We report that CD16(+) cells localize to areas of active inflammation and fibrosis in chronic inflammatory liver disease and that a unique combination of cell surface receptors promotes the transendothelial migration of CD16(+) monocytes through human HSECs under physiological flow. CX(3)CR1 activation was the dominant pertussis-sensitive mechanism controlling transendothelial migration under flow, and expression of the CX(3)CR1 ligand CX(3)CL1 is increased on hepatic sinusoids in chronic inflammatory liver disease. Exposure of CD16(+) monocytes to immobilized purified CX(3)CL1 triggered beta1-integrin-mediated adhesion to vascular cell adhesion molecule-1 and induced the development of a migratory phenotype. Following transmigration or exposure to soluble CX(3)CL1, CD16(+) monocytes rapidly but transiently lost expression of CX(3)CR1. Adhesion and transmigration across HSECs under flow was also dependent on vascular adhesion protein-1 (VAP-1) on the HSECs.

Conclusion: Our data suggest that CD16(+) monocytes are recruited by a combination of adhesive signals involving VAP-1 and CX(3)CR1 mediated integrin-activation. Thus a novel combination of surface molecules, including VAP-1 and CX(3)CL1 promotes the recruitment of CD16(+) monocytes to the liver, allowing them to localize at sites of chronic inflammation and fibrosis.

Figure 1

CD16⁺ and CD16⁻ monocytes were analyzed for the expression of cell adhesion molecules and chemokine receptors. Some surface molecules, such as CX₃CR1, CD49d and CD18 were expressed at the same level on both populations (a, b, e). When compared to CD16⁻ monocytes, molecules such as CD62L and CCR7 were expressed at a lower level or not at all on CD16⁺ monocytes (c, f). CD11c was noted on both populations, but at a slightly higher level on CD16⁺ monocytes (d), consistent with their putative role as mDC precursors. The complete characterization of chemokine receptor expression on three populations of monocytes (h) defined by expression of CD14 and CD16 as CD14⁺CD16⁺, CD14⁻CD16⁺ and CD14⁺CD16⁻ is shown in (g). The plots in (a–f) are representative profiles. The mean ± SEM of five experiments is shown in (g).

Figure 2

Normal and diseased human liver tissue was analysed for the expression of CX₃CL1. (A) Total RNA was analysed for the expression of CX₃CL1 mRNA by qPCR demonstrating a 2-fold increase in primary biliary cirrhosis and a 3–3.5-fold increase in alcoholic liver disease (ALD) and autoimmune hepatitis (AIH) compared with non-inflamed liver. A similar increase was detected in primary sclerosing cholangitis (PSC), though this did not reach significance. All values were relative to normal liver (NL) (n = 6 ± SEM, p ≤ 0.01 *, p ≤ 0.001 **). Immunohistochemical analysis of normal liver (B) revealed expression of CX₃CL1 was limited to biliary structures (arrowed), whilst in diseased liver expression persisted on bile ducts and increased on sinusoids (C).

Figure 3

The localization of CD16⁺ cells was assessed in normal and diseased human liver tissue by immunohistochemistry. In normal liver (A), CD16⁺ cells were located throughout the parenchyma and portal tracts (PT) but in diseased liver (B) were associated with the inflammatory infiltrate and fibrotic septa and largely absent from regenerative nodules (RN).

Figure 4

CD16⁺ monocytes adhere to and transmigrate across HSEC under flow. Pre-treatment of CD16⁺ monocytes with PTX or HSEC with blocking antibodies to VAP-1, ICAM-1 and VCAM-1 or CX₃CL1 resulted in a significant decrease in the total number of cells adhering to the endothelial monolayer from flow (a). We quantified the number of adherent cells that subsequently went on to transmigrate through the HSEC monolayer. Transmigrated cells that have migrated beneath the endothelial monolayer were phase dark and easily distinguished from the phase bright cells adherent on surface of the monolayer. The numbers of adherent cells transmigrating was significantly reduced in the presence of a blocking anti-VAP-1 antibody and virtually abolished by the treatment of HSEC with anti-CX₃CL1 or of CD16⁺ monocytes by pertussis toxin (b). In contrast blocking antibodies to VCAM-1 and ICAM-1 prevented adhesion, but not the transmigration of CD16⁺ monocytes (b). Blocking antibodies to E-Selectin and P-Selectin on HSEC did not affect adhesion or transmigration of CD16⁺ monocytes (not shown), consistent with previous findings that selectins have no role in adhesion in low flow state of liver sinusoids. n = 5 ± p ≤ 0.01 *, p ≤ 0.001 **. HSEC were confirmed to be CD31, L-SIGN, LYVE-1 and VAP-1+ as reported previously, , .

Figure 5

CX₃CL1 treatment induces adhesion and shape change in CD16⁺ monocytes on VCAM-1. CD16⁺ monocytes bound to immobilised VCAM-1 in microslides. Less than 50% of the cells undergo shape change. The combination of immobilised VCAM-1 and CX₃CL1 supported a higher number of adherent cells the majority of which showed morphological changes consistent with a migratory phenotype. Furthermore, analysis of the activation of the α4β1 integrin on CD16⁺ monocytes demonstrated a near doubling of median channel fluorescence following exposure to CX₃CL1 (b). Flow data represent the mean ± SEM of three experiments, cytometry data representative of three repeats. p ≤ 0.001 **

Figure 6

A comparison of CX₃CR1 expression on CD16⁺ monocytes prior to and after transmigration across HSEC in vitro. Purified CD16⁺ monocytes were applied to the top chamber of a transwell coated with HSEC that had been stimulated with TNFα. Transmigrated cells were collected and compared to pre-emigrant cells for CX₃CR1 expression. The transmigrated cells exhibited a near-complete loss of CX₃CR1 expression (a). Additionally, this loss of expression could be recapitulated by incubating CD16⁺ monocytes with CX₃CL1 prior to antibody labelling (b). Pre-incubation for 1hr completely diminished receptor expression which recovered following an additional 1hr rest following removal of exogenous CX₃CL1. We confirmed this was not due to receptor masking by repeating the experiment on ice. This receptor loss following engagement of CX₃CR1 may contribute to retaining precursor DC in the liver promoting their maturation into liver-specific mDC. Representative flow cytometric profiles from three replicate experiments.

Figure 7

A comparison of the expression of CX₃CR1 on CD16⁺ monocytes and mDC from human liver. Peripheral blood CD16⁺ monocytes taken from a patient about to undergo liver transplantation for end stage liver disease exhibited an intermediate level of expression of CX₃CR1, similar to the level of expression noted in healthy donors (Fig 1a). The mDC, representing mature forms of DC precursors that transmigrated across HSEC, were isolated from the liver of the same patient a few hours later and identified by flow cytometry as cells within the monocyte cloud that expressed CD86 and high levels of MHC Class II. The mDC exhibited either high or low expression of CX₃CR1 and when compared to the intermediate level of expression on CD16⁺ monocytes, suggesting that the microenvironment of the liver may regulate the expression of CX₃CR1. Representative flow cytometric profiles from three replicate experiments.