Because recruitment of neutrophils and lymphocytes to the liver involves distinct adhesion pathways,24,

25 we hypothesized that unique combinations of molecules might regulate monocyte recruitment. We report that recruitment of human CD16+ monocytes to the inflamed liver involves unique combinations of adhesion molecules in which interactions mediated by vascular adhesion protein-1 (VAP-1) and the chemokine CX3CL1 are critically important. GPC, G protein-coupled; HSEC, hepatic sinusoidal endothelial cell; ICAM, intercellular adhesion molecule; mAb, monoclonal antibody; mDC, myeloid dendritic cell; PBS, phosphate-buffered saline; PTX, pertussis toxin; TNF-α, tumor necrosis factor-α; VAP-1, vascular

adhesion protein-1; VCAM, vascular cell adhesion molecule. selleck products Liver tissue was obtained from livers removed at transplantation at the Queen Elizabeth Hospital from patients with alcoholic liver disease (n = 6), primary biliary cirrhosis (n = 6), primary sclerosing cholangitis (n = 6), and autoimmune hepatitis (n = 6). Peripheral Ivacaftor molecular weight blood was obtained from healthy volunteers and liver transplant recipients. Samples were collected after informed consent following local Ethics Committee approval. Soluble CX3CL1 and all anti-chemokine receptor monoclonal antibodies (mAbs) except anti-CX3CR1 were obtained from R&D Systems Europe and used at the recommended concentrations (Table 1). Six-micrometer cryostat sections were air-dried on poly-L-lysine treated slides, acetone-fixed (10 minutes), and stained. Sections were preincubated with 2.5% horse serum (Vector Laboratories, Peterborough, UK) in TBS prior to mouse anti-human mAb against CD16 or CX3CL1 in Tris-buffered saline/0.1% normal horse

serum. Control sections were incubated with isotype-matched control mAb. Antibody binding was assessed using ImmPress peroxidise 上海皓元医药股份有限公司 visualisation with Novared chromogen (Vector Laboratories). Sections were counterstained with hematoxylin. Total RNA was extracted from 30 mg human liver using RNEasy (Qiagen, UK) after DNAse treatment with RNAse-free DNAse (Qiagen). Fifty micrograms extracted RNA was transcribed into complementary DNA using iScript cDNA (BioRad, Hercules, CA), and eluted RNA and complementary DNA were measured (NanoDrop, Thermo-Fisher Scientific). Expression of human CX3CL1 messenger RNA was quantified using Taqman Fluorogenic 5′-nuclease assays and gene-specific 5′-FAM-labeled probes on an ABI Prism 7900 detector. Threshold cycle (Ct) values of the target gene were normalized to GAPDH and differential expression levels calculated using 2−ΔΔCt. Blood mononuclear cells isolated using Lympholyte (Cedarlane Laboratories, Burlington, Canada) were resuspended in labelling medium (phosphate-buffered saline [PBS]/0.5% fetal bovine serum/0.1 mM ethylene diamine tetraacetic acid).