SCARF-1 promotes adhesion of CD4+ T cells to human hepatic sinusoidal endothelium under conditions of shear stress

Abstract

Liver-resident cells are constantly exposed to gut-derived antigens via portal blood and, as a consequence, they express a unique repertoire of scavenger receptors. Whilst there is increasing evidence that the gut contributes to chronic inflammatory liver disease, the role of scavenger receptors in regulating liver inflammation remains limited. Here, we describe for the first time the expression of scavenger receptor class F, member 1 (SCARF-1) on hepatic sinusoidal endothelial cells (HSEC). We report that SCARF-1 shows a highly localised expression pattern and co-localised with endothelial markers on sinusoidal endothelium. Analysis of chronically inflamed liver tissue demonstrated accumulation of SCARF-1 at sites of CD4⁺ T cell aggregation. We then studied the regulation and functional role of SCARF-1 in HSEC and showed that SCARF-1 expression by HSEC is regulated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS). Furthermore, SCARF-1 expression by HSEC, induced by proinflammatory and gut-derived factors acts as a novel adhesion molecule, present in adhesive cup structures, that specifically supports CD4⁺ T cells under conditions of physiological shear stress. In conclusion, we show that SCARF-1 contributes to lymphocyte subset adhesion to primary human HSEC and could play an important role in regulating the inflammatory response during chronic liver disease.

Figure 1

SCARF-1 expression is up-regulated in chronic liver disease. (a) Immunohistochemical staining of SCARF-1 (brown) in representative images of normal liver (NL), primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), alcoholic liver disease (ALD) and non-alcoholic steatohepatitis (NASH). Insets show a higher magnification of the parenchymal tissues. Fibrotic septa are delineated by the dashed black lines. Scale bar = 200 µm. Inset scale bar = 50 µm. Surface area quantification of immunohistochemical staining. ****Represents statistical significance where p ≤ 0.001. n = 4–8 in each group (bottom right panel). (b) Western blot (left panel) and quantification (right panels) of the ~180 kDa, ~90 kDa and ~60 kDa species of SCARF-1 in normal liver (NL) and chronic liver disease (CLD). **Represents statistical significance where p ≤ 0.01. n = 4–8 in each group. Results are regions cropped from the same membrane (see Supplementary Figure 7). (c) Sandwich ELISA analysis of soluble SCARF-1 (sSCARF-1) in human serum. n = 5–10 in each group. (d) Western blot of SCARF-1 in serum from 3 individual PSC patients. Black arrow indicates the major species at ~60 kDa. Results cropped from the same membrane (see Supplementary Figure 7). (e) SCARF-1 mRNA expression in normal liver (NL) and chronic liver disease (CLD) tissue. ***Represents p ≤ 0.005. n = 8 in NL and n = 26 in CLD group.

Figure 2

HSEC represent a major cell type expressing SCARF-1 in CLD. (a) Representative images of dual colour immunofluorescent staining on chronically diseased (ALD and PSC) liver for SCARF-1 (green) and endothelial marker CD31 (top left panel), activated stellate cell marker α-SMA (smooth muscle actin; top middle panel), fibroblast marker CD90 (top right panel), macrophage marker CD68 (bottom left panel), hepatocyte marker CK18 (bottom middle panel) and biliary epithelial marker EpCAM (bottom right panel). Insets show magnification of SCARF-1 and cell-specific markers. Scale bar = 50 µm. Inset scale bar = 10 µm. (b) SCARF-1 mRNA expression in isolated human hepatic sinusoidal endothelial cells (HSECs), hepatic stellate cells (HSCs) and activated liver myofibroblasts (aLMFs). ****Indicates statistical significance where p ≤ 0.001. n = 5–10 in each group. (c) Representative images of dual colour immunofluorescent staining of SCARF-1 (green) and CD4 (red) in normal liver (NL) and chronically diseased livers (PSC and PBC). Scale bar = 250 µm. White dashed lines delineate sites of intensity measurements. (b) Intensity measurements of immunofluorescent staining shown in (a). Black arrows indicate areas of stronf co-localisation of SCARF-1 (green) and CD4 (red).

Figure 3

In vitro expression of SCARF-1 in HSEC can be up-regulated by proinflammatory cytokines and LPS. (a) Representative images of immunofluorescent staining of SCARF-1 (green) with DAPI nuclear stain (blue). Scale bar = 25 µm. (b) Representative Western blot (left panel) and quantification (right panels) of the 180 kDa (dimeric) and 90 kDa (monomeric) species of SCARF-1 in stimulated HSEC compared to media alone control (Con). Results are representative of 3 independent experiments and are regions cropped from the same membrane (see Supplementary Figure 7). (c) Fold change in SCARF-1 protein expression measured by cell-based ELISA in stimulated HSEC. (d) qPCR analysis of SCARF-1 mRNA in stimulated HSEC. (a–d) HSEC were treated with 10 ng/ml of tumour necrosis factor (TNF)α, 10 ng/ml of interferon (IFN)γ, or both in combination or with 1 µg/ml of LPS for 24 h. (c and d) Dotted lines indicate control level of expression. * and ** indicate statistical significance where p ≤ 0.05 and p ≤ 0.01, respectively. (b and d) n = 3 and (c) n = 5 independent experiments with different HSEC donors in each group.

Figure 4

Recombinant human (rh)SCARF-1 mediates the adherence of CD4⁺ T lymphocytes in the presence of rhVCAM-1, under conditions of flow. (a) A schematic representation of the immobilised protein flow assay. (b and c) Quantification of CD4⁺ and CD8⁺ T cells adhered to immobilised rhVCAM-1 (10 µg/ml) and VCAM-1 in the presence of rhSCARF-1 (10 µg/ml). (d and e) Quantification of CD4⁺ and CD8⁺ T cells adhered to immobilised rhVCAM-1 and rhSCARF-1 pre-treated with isotype matched control (IMC; 10 µg/ml) or SCARF-1 blocking antibody (10 µg/ml). ** and ****indicate statistical significance where p ≤ 0.01 and p ≤ 0.001, respectively. n = 3 independent experiments with different lymphocyte donors, with 12 fields of view taken from each.

Figure 5

SCARF-1 forms an adhesive cup for CD4⁺ T cell adherence to HSEC. (a–d) Representative image of CD4⁺ T cells (blue; Cell Trace Violet (CTV)-labelled) attached to TNFα-stimulated HSEC (green; Cell Tracker Green (CTG)-labelled) via SCARF-1 (red) adhesive cups, which are rich in (c) filamentous (F-)actin (pink) and (d) ICAM-1 (grey). (a) White arrows highlight adherent CD4⁺ T lymphocytes. Scale bar = 40 µm. (b) White dotted line represents the periphery of the HSEC and the white dashed line delineates the site of the Z stack. Scale bars = 12 µm (left) and 2 µm (right). (c) White arrow highlights adherent CD4⁺ T lymphocyte. Scale bars = 22.5 µm (top left) and 7 µm (right). (d) White arrows highlight adherent CD4⁺ T lymphocytes. Scale bars = 50 µm (top left) and 5 µm (right). (c and d) White asterisks indicate adherent CD4⁺ T lymphocyte in adhesive cup.

Figure 6

Antibody blockade of SCARF-1 on HSEC inhibits the adherence of CD4⁺ T lymphocytes in the presence of TNFα and LPS. (a,b and c; left panels) Representative images of CD4⁺ T cells adhered to HSEC stimulated with (a) TNFα (10 ng/ml), (b) LPS (1 µg/ml) or (c) TNFα and LPS together. HSEC were pre-treated with isotype matched controls (IMC; 10 µg/ml), SCARF-1 blocking antibody (10 µg/ml), VCAM-1 blocking antibody (10 µg/ml) or SCARF-1 and VCAM-1 in combination (both 10 µg/ml). Black arrowheads highlight adherent CD4⁺ T lymphocytes on the HSEC monolayer. (a,b and c; right panels) Quantification of adherent CD4⁺ T cells in the presence of the blocking antibodies, with the percentage inhibition indicated. *, **, *** and **** all indicate statistical significance where p ≤ 0.05, p ≤ 0.01, p ≤ 0.005 and p ≤ 0.001, respectively. ns = not significant. n = 3 independent experiments with different lymphocyte and HSEC donors, with 12 fields of view taken from each.

Figure 7

siRNA knockdown of SCARF-1 in HSEC significantly decreases CD4⁺ T cell adherence. (a) Representative Western blot analysis of SCARF-1 ~180 kDa and ~90 kDa species in HSEC treated with control and SCARF-1 siRNA knockdown. Results are regions cropped from the same membrane (see Supplementary Figure 7) (b) Quantification of SCARF-1 expression in HSEC treated with siRNA knockdown of SCARF-1 expressed as a % of expression in control HSEC. n = 3 independent experiments with different HSEC. *Indicates statistical significance where p ≤ 0.05. (c) Quantification of adherence of lymphocytes to monolayers of HSEC in flow assays pre-treated with control siRNA and SCARF-1 siRNA. n = 3 independent experiments with different HSEC donors, with 12 fields of view taken from each. ****Indicates statistical significance where p ≤ 0.001.