Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration

Abstract

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs.

Conclusion: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294-309).

Figure 1

Lymphocytes migrate into sinusoidal endothelial cells in human chronic liver disease. (A) Immunofluorescent staining of liver sections from a patient with primary biliary cirrhosis demonstrating the presence of lymphocytes in portal regions within fibrous septum. (B) Immunofluorescent staining of the same liver demonstrating lymphocytes within the sinusoidal channels. CD3+ lymphocytes appear in red and stabilin‐1–positive hepatic sinusoidal endothelium appears in green. (C) Lymphocytes (red) within the sinusoidal channels with one lymphocyte within the sinusoidal lumen and another within the endothelial cell (green). Arrows indicate the intraendothelial lymphocyte. (D) Two‐dimensional image of orthogonal (XZ) projection of a CD3+ lymphocyte (red) colocalizing with a stabilin‐1–positive (green) endothelial cell. (E,F) Three‐dimensional reconstruction of the orthogonal (XZ) projection in panel D with CD3+ red signal only (E) and overlay of stabilin‐1–positive green signal (F). Scale bars = 20 μm (A,B), 10 μm (C), and 5 μm (D).

Figure 2

Intracellular migration of lymphocytes into primary HSECs. (A) Representative confocal images of lymphocytes adherent to cytokine‐treated HSEC monolayer. HSEC cytoplasm was stained with CellTracker CMFDA (green), lymphocyte membrane was stained with CD4 marker (red), and HSEC and lymphocyte nuclei were stained with DAPI (blue). (B) Immunofluorescent staining for lysosomal markers VAMP‐7 (red) and (C) CD63 (red) were performed. Orthogonal (XZ) projections are shown corresponding to the plane of the red line in the overlay images. Arrows in the orthogonal projections indicate the HSEC/lymphocyte nucleus and its relationship to the lysosomal compartment. (D) Quantification of intracellular migration. (E) Adhesion of peripheral blood lymphocytes on cytokine‐treated HSECs. (F) Quantification of intracellular migration on TNFα‐ and IFNγ‐treated HSECs at various time points. Quantitative data are the mean ± SEM of three independent experiments. Statistical significance was determined using one‐way analysis of variance, with a Tukey's post hoc multiple comparison test. *P < 0.05. Scale bars = 20 μm (A), 10 μm (B,C), and 5 μm (A‐C, orthogonal projections).

Figure 3

Nonviable lymphocytes are not internalized by HSECs. (A) Representative confocal images of nonviable (fixed) lymphocytes perfused over TNFα‐ and IFNγ‐treated HSECs in a flow adhesion assay. Endothelial cells were stained with CellTracker CFMDA (green) and nuclei were stained with DAPI (blue). (B) Repeat experiments with viable lymphocytes. Arrows indicate intracellular lymphocytes. Images are representative of three independent experiments. Scale bar = 30 μm (A,B).

Figure 4

IFNγ promotes intracellular migration of lymphocytes into HSECs but not HUVECs. (A,B) Representative confocal images of lymphocytes adherent to TNFα‐ and IFNγ‐treated HSEC and HUVEC monolayers in a flow adhesion assay. Endothelial cells were stained with CellTracker CFMDA (green) and nuclei were stained with DAPI (blue). Arrows indicate intracellular lymphocytes. (C) Quantification of adhesion and (D) intracellular migration of lymphocytes into HSEC and HUVEC monolayers in a flow adhesion assay. Quantitative data are the mean ± SEM of five independent experiments. Statistical significance was determined using a two‐tailed t test. *P < 0.05. Scale bars = 25 μm (A,B).

Figure 5

Intracellular migration is dependent on ICAM‐1 but not inhibited by disruption of junctional complexes. (A) Quantification of intracellular migration of lymphocytes across HSECs pretreated with blocking antibodies to ICAM‐1, CLEVER‐1/stabilin‐1 (STAB‐1), and PDL1. Results are the mean ± SEM of at least three independent experiments. (B) Quantification of intracellular migration of lymphocytes pretreated with CXCR3 blocking antibody or IMC across HSECs. Results are the mean ± SEM of at least three independent experiments. (C) Quantification of intracellular migration of lymphocytes across HSECs pretreated with control media or media supplemented with Blebbistatin (Blebb) or cytochalasin D (Cyto D). Results are the mean ± SEM of three independent experiments. (D) Measurement of transelectrical endothelial resistance across untreated or cytokine‐treated HSECs and HUVECs. Results are the mean ± SEM of six independent experiments. (E) Representative images of cell tracker CFMDA‐labeled endothelial monolayers (green) after flow assay pretreated with TNFα and IFNγ and cultured in flow media or calcium‐free flow media. Arrows indicate lymphocyte intracellular migration. (F) Quantification of intracellular migration of lymphocytes across cytokine‐treated HSEC and HUVEC monolayers cultured in flow media (Media) or calcium‐free flow media. Results are the mean ± SEM of three independent experiments. Statistical significance was determined using a two‐tailed t test (A‐C) and one‐way analysis of variance with a Tukey's post hoc multiple comparison test (D,F). Scale bar = 25 μm (E). *P < 0.05. **P < 0.005. ****P < 0.0005.

Figure 6

Intracellular crawling of lymphocytes across HSEC monolayers. (A) Live cell imaging of peripheral blood lymphocytes migrating across TNFα‐ and IFNγ‐treated HSECs under shear stress. HSECs were stained with CellTracker CFMDA (green), lymphocytes with cell tracker BMQC (blue), and HSEC junctions with CellMask orange plasma membrane stain (red). Orthogonal (XZ) projection is shown corresponding to the plane of the red line. (B) Left: Representative transmission electron microscopy image of an intracellular lymphocyte within TNFα‐ and IFNγ‐treated HSECs. Right: Arrows in high magnification images of two regions within the intracellular lymphocyte indicate a double membrane. (C) Still images of http://onlinelibrary.wiley.com/doi/10.1002/hep.28879/suppinfo taken at 1‐minute intervals of time‐lapse recordings of lymphocytes migrating across TNFα‐ and IFNγ‐stimulated HSEC monolayer under shear stress. HSEC cytoplasm was prelabeled with CellTracker CFMDA (green) and lymphocytes were prelabeled with CellTracker BMQC (red). (D) The same sequence of images shown in panel C with the red (lymphocyte) signal omitted. The arrows indicate lymphocytes (red) undergoing intracellular crawling from one endothelial cell to the adjacent cell displacing the cytoplasm of the endothelial cell (green). Note the redistribution of endothelial cytoplasm in panel D as the lymphocytes migrate from cell to cell. (E) Representative confocal images of lymphocyte cell‐to‐cell crawling across TNFα‐ and IFNγ‐treated HSEC monolayer. HSEC cytoplasm was stained with CellTracker CFMDA (green) and HSECs and lymphocyte nuclei were stained with DAPI (blue) and VE‐cadherin (red). Arrows indicate disruption of VE‐cadherin at the endothelial junction as lymphocytes migrate to the adjacent HSECs. (F) Representative confocal images of experiment with same conditions as panel D with staining of CD31 (red). Arrows indicate enrichment of CD31 at the junction as lymphocyte migrates to the adjacent HSECs. Scale bars = 25 μm (A,C,D), 10 μm (A, orthogonal projection, E,F), 2 μm (B, left), and 1 μm (B, right).

Figure 7

Junctional molecular expression differs between HSECs and HUVECs. (A‐D) Immunofluorescent staining of primary HSECs for CLEVER‐1/stabilin‐1 (green) and junctional molecules (red). Images representative of three separate HSEC isolates. (E) Cell‐based ELISA of junctional molecule expression in HSECs isolated from normal livers and chronically inflamed livers. (F) Cell‐based ELISA of junctional molecule expression in HSECs compared with HUVECs. Data are the mean of three experiments and values represent the mean optical density at 490 nm of three replicate wells minus the optical density of an isotype‐matched control antibody. Statistical significance was determined by two‐tailed t test. **P < 0.005. Scale bars = 20 μm (A‐D).

Figure 8

Microarray demonstrates differential gene expression changes between HSECs and HUVECs in response to TNFα and IFNγ challenge. (A) Heat map images of gene expression changes. Unstimulated HSECs and HUVECs were compared with HSECs and HUVECs that had been stimulated with TNFα and IFNγ (10 ng/mL) for 24 hours. (B) Summary of the total number of mutual and exclusively up‐regulated and down‐regulated genes after cytokine stimulation in HSECs and HUVECs. Genes were identified to be up‐regulated or down‐regulated based on log ratios that were 2‐fold or greater. (C) Comparative messenger RNA expression between HUVECs and HSECs of occludin, CD36, macrophage mannose receptor (MRC‐1), and podoplanin (PDPN). (D,E) Pathway analysis of up‐regulated and down‐regulated genes in unstimulated HSECs compared with HUVECs. (F) Pathway analysis of down‐regulated genes in stimulated HUVECs compared with stimulated HSECs. The pathways are plotted against their corresponding −log10 values of probability on the x axis. Statistical significance was determined using a two‐tailed t test. ***P < 0.001. ****P < 0.0005.