CXCR3 Activation Promotes Lymphocyte Transendothelial Migration across Human Hepatic Endothelium under Fluid Flow
2005; Elsevier BV; Volume: 167; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)62060-3
ISSN1525-2191
AutoresStuart M. Curbishley, Bertus Eksteen, Ron P. Gladue, Patricia F. Lalor, David H. Adams,
Tópico(s)Liver physiology and pathology
ResumoT cells infiltrating the inflamed liver express high levels of CXCR3 and show enhanced migration to CXCR3 ligands in chemotactic assays. Moreover, CXCR3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR3high lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR3 ligands secreted by the endothelium or by exogenous CXCR3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver. T cells infiltrating the inflamed liver express high levels of CXCR3 and show enhanced migration to CXCR3 ligands in chemotactic assays. Moreover, CXCR3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR3high lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR3 ligands secreted by the endothelium or by exogenous CXCR3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver. Chronic inflammation occurs as a consequence of the recruitment and retention of lymphocytes in tissue. The recruitment of effector lymphocytes from the circulation is dependent on interactions between lymphocytes and specific cell surface molecules expressed on endothelial cells. Once captured the retention and positioning of leukocytes within tissue requires signals to localize and retain them at sites of target cell damage. These receptors and chemokine signals can be organized into the accepted multistep paradigm of leukocyte adhesion to vascular endothelium, which is relevant to most organ systems, although the specific signals involved differ between tissues.1Campbell JJ Butcher EC Chemokines in tissue-specific and microenvironment-specific lymphocyte homing.Curr Opin Immunol. 2000; 12: 336-341Crossref PubMed Scopus (560) Google Scholar, 2von Andrian UH Mackay CR T-cell function and migration. Two sides of the same coin.N Engl J Med. 2000; 343: 1020-1034Crossref PubMed Scopus (1242) Google Scholar Initial transient interactions between flowing lymphocytes and endothelium tether the lymphocyte and induce it to roll on the vessel wall where it comes into contact with chemokines in the endothelial glycocalyx. In the presence of an appropriate chemokine, specific G-protein-coupled receptors on the lymphocyte are activated triggering high-affinity β1 and β2 integrins on the leukocyte surface to bind endothelial ligands resulting in arrest and stable adhesion.3Pachynski RK Wu SW Gunn MD Erle DJ Secondary lymphoid-tissue chemokine (SLC) stimulates integrin alpha 4 beta 7-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow.J Immunol. 1998; 161: 952-956PubMed Google Scholar This is followed by shape change of the lymphocyte associated with migration on and through the endothelium and into underlying tissue where cells accumulate at the focus of inflammation, a process driven by chemotactic signals.4Foxman EF Kunkel EJ Butcher EC Integrating conflicting chemotactic signals. The role of memory in leukocyte navigation.J Cell Biol. 1999; 147: 577-588Crossref PubMed Scopus (193) Google Scholar, 5Foxman EF Campbell JJ Butcher EC Multistep navigation and the combinatorial control of leukocyte chemotaxis.J Cell Biol. 1997; 139: 1349-1360Crossref PubMed Scopus (451) Google Scholar Chemokines are critical components of this adhesion cascade and are believed to play two crucial roles: triggering integrin-mediated stable adhesion and directing migration. Chemokines can bind to endothelial glycosaminoglycans allowing them to be presented to flowing leukocytes and also providing a mechanism for the paracrine presentation of chemokines secreted by other cells within the microenvironment, a process termed “posting”.6Tanaka Y Adams DH Shaw S Proteoglycan on endothelial cells present adhesion-inducing cytokines to leukocytes.Immunol Today. 1993; 14: 111-114Abstract Full Text PDF PubMed Scopus (391) Google Scholar, 7Gretz JE Kaldjian EP Anderson AO Shaw S Sophisticated strategies for information encounter in the lymph node: the reticular network as a conduit of soluble information and a highway for cell traffic.J Immunol. 1996; 157: 495-499Crossref PubMed Google Scholar, 8Ebnet K Kaldjian EP Anderson AO Shaw S Orchestrated information-transfer underlying leukocyte-endothelial interactions.Annu Rev Immunol. 1996; 14: 155-177Crossref PubMed Scopus (184) Google Scholar, 9Tanaka Y Adams DH Hubscher S Hirano H Siebenlist U Shaw S T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1β.Nature. 1993; 361: 79-82Crossref PubMed Scopus (875) Google Scholar, 10Middleton J Patterson AM Gardner L Schmutz C Ashton BA Leukocyte extravasation: chemokine transport and presentation by the endothelium.Blood. 2002; 100: 3853-3860Crossref PubMed Scopus (397) Google Scholar, 11Middleton J Neil S Wintle J Clark-Lewis I Moore H Lam C Auer M Hub E Rot A Transcytosis and surface presentation of IL-8 by venular endothelial cells.Cell. 1997; 91: 385-395Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar Similar mechanisms are believed to be involved in both normal immune surveillance and in inflammatory disease although the chemokines involved differ with constitutive chemokines playing the dominant role in physiological trafficking and inducible inflammatory cytokines involved in inflammation.1Campbell JJ Butcher EC Chemokines in tissue-specific and microenvironment-specific lymphocyte homing.Curr Opin Immunol. 2000; 12: 336-341Crossref PubMed Scopus (560) Google Scholar Chemokines can be classified into four groups according to their amino acid sequence.12Murphy PM Baggiolini M Charo IF Hebert CA Horuk R Matsushima K Miller LH Oppenheim JJ Power CA International Union of Pharmacology. XXII. Nomenclature for chemokine receptors.Pharmacol Rev. 2000; 52: 145-176PubMed Google Scholar The two largest groups are the CC chemokines, where conserved cysteine residues lie adjacent to each other, and CXC chemokines where an amino acid separates the first two cysteine residues. This group can be further classified by the presence of a glutamic acid-leucine-arginine (ELR) sequence near the N terminus. The ELR-containing CXC chemokines are potent chemoattractants for neutrophils and include interleukin (IL)-8.13Larsen CG Anderson AO Appella E Oppenheim JJ Matsushima K The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes.Science. 1989; 243: 1464-1466Crossref PubMed Scopus (998) Google Scholar CXC chemokines lacking the ELR motif are CXCL10 or interferon (IFN)-inducible protein (IP-10), CXCL9 or monokine induced by IFN-γ (MIG), and CXCL11 or IFN-inducible T-cell α-chemoattractant (ITAC), all of which display potent lymphocyte chemotactic activity.14Meyer M Hensbergen PJ van der Raaij-Helmer EM Brandacher G Margreiter R Heufler C Koch F Narumi S Werner ER Colvin R Luster AD Tensen CP Werner-Felmayer G Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection.Eur J Immunol. 2001; 31: 2521-2527Crossref PubMed Scopus (57) Google Scholar, 15Cole KE Strick CA Paradis TJ Ogborne KT Loetscher M Gladue RP Lin W Boyd JG Moser B Wood DE Sahagan BG Neote K Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.J Exp Med. 1998; 187: 2009-2021Crossref PubMed Scopus (749) Google Scholar, 16Taub DD Longo DL Murphy WJ Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T-lymphocytes into the peripheral-tissues of human peripheral-blood lymphocytes-SCID mice.Blood. 1996; 87: 1423-1431PubMed Google Scholar, 17Farber JM HuMig: a new human member of the chemokine family of cytokines.Biochem Biophys Res Commun. 1993; 192: 223-230Crossref PubMed Scopus (167) Google Scholar These three chemokines bind a common receptor, CXCR3, the expression of which is increased on tissue-infiltrating T cells and Th1-polarized T cells. The observation that CXCR3 ligands require IFN-γ for their expression and are localized to sites of inflammation led to the assumption that CXCR3 is important for effector lymphocyte recruitment into inflamed tissue. Studies using CXCR3-deficient or IP-10-deficient mice show reduced tissue infiltration of effector cells in several inflammatory and transplantation models18Dufour JH Dziejman M Liu MT Leung JH Lane TE Luster AD IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking.J Immunol. 2002; 168: 3195-3204PubMed Google Scholar, 19Hancock WW Lu B Gao W Csizmadia V Faia K King JA Smiley ST Ling M Gerard NP Gerard C Requirement of the chemokine re-ceptor CXCR3 for acute allograft rejection.J Exp Med. 2000; 192: 1515-1520Crossref PubMed Scopus (570) Google Scholar supporting the hypothesis but the role played by CXCR3 remains unclear. CXCR3 ligands have been shown to promote adhesion of lymphoblasts to human umbilical vein endothelial cells in vitro20Mohan K Ding Z Hanly J Issekutz TB IFN-gamma-inducible T cell alpha chemoattractant is a potent stimulator of normal human blood T lymphocyte transendothelial migration: differential regulation by IFN-gamma and TNF-alpha.J Immunol. 2002; 168: 6420-6428PubMed Google Scholar but have not previously been shown to be involved in transendothelial migration under conditions of physiological flow. We have previously reported that T cells infiltrating the inflamed liver express high levels of CXCR3 and show enhanced migration to CXCR3 ligands in chemotactic assays.21Yoong KF Afford SC Jones R Aujla P Qin S Price K Hubscher SG Adams DH Expression and function of CXC and CC chemokines in human malignant liver tumors: a role for human monokine induced by gamma-interferon in lymphocyte recruitment to hepatocellular carcinoma.Hepatology. 1999; 30: 100-111Crossref PubMed Scopus (129) Google Scholar, 22Shields PL Morland CM Salmon M Qin S Hubscher SG Adams DH Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver.J Immunol. 1999; 163: 6236-6243PubMed Google Scholar, 23Goddard S Williams A Morland C Qin S Gladue R Hubscher SG Adams DH Differential expression of chemokines and chemokine receptors shapes the inflammatory response in rejecting human liver transplants.Transplantation. 2001; 72: 1957-1967Crossref PubMed Scopus (125) Google Scholar Moreover CXCR3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis and their presence has been correlated with outcome of inflammatory liver disease.24Arai K Liu ZX Lane T Dennert G IP-10 and Mig facilitate accumulation of T cells in the virus-infected liver.Cell Immunol. 2002; 219: 48-56Crossref PubMed Scopus (54) Google Scholar, 25Itoh Y Morita A Nishioji K Narumi S Toyama T Daimon Y Nakamura H Kirishima T Okanoue T Clinical significance of elevated serum interferon-inducible protein-10 levels in hepatitis C virus carriers with persistently normal serum transaminase levels.J Viral Hepatol. 2001; 8: 341-348Crossref PubMed Scopus (52) Google Scholar, 26Harvey CE Post JJ Palladinetti P Freeman AJ French RA Kumar RK Marinos G Lloyd AR Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation.J Leukoc Biol. 2003; 74: 360-369Crossref PubMed Scopus (222) Google Scholar, 27Narumi S Tominaga Y Tamaru M Shimai S Okumura H Nishioji K Itoh Y Okanoue T Expression of IFN-inducible protein-10 in chronic hepatitis.J Immunol. 1997; 158: 5536-5544PubMed Google Scholar We have developed a flow-based adhesion assay using human hepatic endothelium which allows us to model leukocyte interactions with hepatic endothelium under physiological conditions of blood flow.28Lalor PF Edwards S McNab G Salmi M Jalkanen S Adams DH Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.J Immunol. 2002; 169: 983-992PubMed Google Scholar In the present study we used this assay to investigate the function of CXCR3 ligands in promoting lymphocyte adhesion and transendothelial migration. To more accurately model the function of in vivo-activated CXCR3high lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR3 not only promotes the adhesion of effector T cells to endothelium from flow but also drives transendothelial migration. Moreover, these responses can be stimulated either by endogenous CXCR3 ligands secreted by the endothelium or by exogenous CXCR3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates the ability of either endogenously or exogenously secreted CXCR3 ligands on human hepatic endothelium to promote lymphocyte adhesion and transendothelial migration under flow. Liver tissue and peripheral blood samples were obtained during liver transplantation at the Liver Unit at the Queen Elizabeth Hospital, Edgbaston, Birmingham, UK. All samples were collected with appropriate patient consent and local ethical committee approval. Six-μm cryostat sections of 1-cm3 liver blocks were cut for immunohistochemistry and immunofluorescence. These sections were air-dried on slides pretreated with Fro-tissue pen (The Binding Site, Birmingham, UK), then fixed for 10 minutes in acetone before staining. Tissue sections were initially incubated with avidin/biotin blocking kit (DAKO, Ely, UK) and washed in Tris-buffered saline (TBS) buffer. Subsequently, the sections were incubated with 20% normal goat serum in TBS buffer for 30 minutes before the addition of a mouse anti-human monoclonal primary antibody (CXCL9: 49106, 5 μg/ml; CXCL10: 6D4, 2 μg/ml; CXCL11: 87328, 5 μg/ml; all from R&D Systems Europe, Abingdon, UK) in TBS for 60 minutes at 37°C in a humidified container. Control sections were incubated without the primary antibody or relevant isotype-matched control. Primary antibody binding was assessed using the StreptABComplex Duet peroxidase kit (DAKO) and developed with diaminobenzidine chromogen (Sigma FastDAB with 1 mg/L of sodium azide; Sigma, Poole, UK) for 5 minutes. The sections were washed in distilled water, counterstained with hematoxylin, and mounted. Positive staining was identified by the presence of a dark brown reaction product. All washes were performed with Tris-buffered saline, pH 7.6. Tissue sections were initially incubated with 20% normal goat serum in TBS buffer for 30 minutes before the addition of both mouse anti-human monoclonal CXCL10 IgG2a (6D4, 2 μg/ml) and CD31 IgG1 (JC70A, 5 μg/ml; DAKO) antibodies in TBS for 60 minutes at 37°C in a humidified container. The slides were then washed in TBS buffer and incubated with goat anti-mouse fluorescein isothiocyanate (IgG2a) and TXR (IgG1) (both Southern Biotechnology, Birmingham, AL) in TBS for 60 minutes at 37°C and protected from light. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma) and the sections mounted with fluorescence mounting medium (DAKO). Dual immunofluorescence was assessed using AxioVision software (Zeiss, Germany). Lymphocytes were isolated from a variety of diseased livers (hepatitis C virus, primary biliary cirrhosis, alcoholic liver disease, primary sclerosing cholangitis) using nonenzymatic, mechanical methods to preserve chemokine receptor expression. Resected tissue was reduced to 5-mm3 cubes and placed for 5 minutes at 230rpm in a Stomacher 400 circulator (Seward, UK). The resultant homogenized tissue was filtered through a fine gauze mesh and lymphocytes were separated by layering over a 33%/77% Percoll (Amersham Biosciences, UK) density gradient and centrifugation at 650 × g for 30 minutes. Isolated lymphocytes were resuspended in RPMI 1640 (Invitrogen, Paisley, UK) with 10% fetal calf serum at 4°C. Peripheral blood lymphocytes were isolated as previously described29Lalor PF Clements JM Pigott R Humphries MJ Spragg JH Nash GB Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.Eur J Immunol. 1997; 27: 1422-1426Crossref PubMed Scopus (37) Google Scholar by density gradient centrifugation over Lymphoprep (Nycomed Pharma, Norway) for 30 minutes at 650 × g. Harvested lymphocytes were washed and resuspended in RPMI 1640/10% fetal calf serum at 4°C before use. Lymphocytes were resuspended in cold fluorescence-activated cell sorting media consisting of 0.5 mmol/L MgCl2, 1.0 mmol/L CaCl2, phosphate-buffered saline (Sigma), pH 7.4, with 10% fetal calf serum and 1 mg/ml sodium azide. Cells were subsequently blocked with 3 mg/ml of mouse immunoglobulins (Sigma) and labeled with the addition of three directly conjugated primary monoclonal antibodies in relevant combinations [CXCR3-RPE: 49801; 2.5 μg/ml (R&D Systems); CD3-RPE-Cy5: UCHT1; 1:10, CD8-fluorescein isothiocyanate: DK25; 1:10 (both DAKO); CD4-ECD: SFCI12T4D11; 1:10, CD3-ECD: HIT3a; 1:10 (both Beckman Coulter UK Ltd.); CD45RA-RPE-Cy5: MEM56; 1:20, CD45R0-fluorescein isothiocyanate: UCHL1; 10 μg/ml (both Serotec, Oxford, UK)]. Irrelevant isotype-matched antibodies were used to label control samples. Cells were fixed in 1% paraformaldehyde before three-color analysis on a Coulter Epics XL flow cytometer (Coulter Electronics Ltd., UK). Results were analyzed on Summit software (Dako Cytomation, UK). HSECs and BECs were isolated according to our previously described methods.28Lalor PF Edwards S McNab G Salmi M Jalkanen S Adams DH Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.J Immunol. 2002; 169: 983-992PubMed Google Scholar, 30Joplin R Isolation and culture of biliary epithelial cells.Gut. 1994; 35: 875-878Crossref PubMed Scopus (34) Google Scholar Briefly, ∼30 g of liver tissue was removed from explanted livers of patients undergoing transplantation. The tissue was finely chopped and subjected to enzymatic digestion (1 mg/ml, collagenase type IV; Sigma) and metrizamide density gradient centrifugation (25%, w/v; Sigma). The nonparenchymal cell band was then removed and cells were further purified by immunomagnetic selection. Cells positive for mAb HEA-125 (Progen, Germany) were classified BECs, whereas those cells negative for HEA-125 but positive for CD31 (DAKO) were classified HSECs. After isolation HSECs and BECs were plated in collagen-coated 25-cm2 tissue culture flasks (Corning). HSECs were cultured in human endothelial basal media (PAA Laboratories, Somerset, UK) containing 10% human serum (H&D Supplies, Bucks, UK), penicillin, and streptomycin (100 U/ml), hepatocyte growth factor (5 ng/ml, PromoCell) and vascular endothelial growth factor (10 ng/ml, R&D Systems). BECs were cultured in Dulbecco's modified Eagle's medium, Ham's F12 (Invitrogen), containing 10% human serum, penicillin, and streptomycin (100 IU/ml), glutamine (2 mmol/L), epidermal growth factor (10 ng/ml, R&D Systems), hydrocortisone (2 mg/ml), cholera toxin (10 ng/ml), triiodothyronine (2 nmol/L), all Sigma, insulin (0.124 U/ml, Novo Nordisk, W. Sussex, UK), hepatocyte growth factor (5 ng/ml). Cells were cultured to confluence and in all experiments used between passages 2 and 6. Cells were plated at an initial count of 1 × 105/ml and grown to confluence in collagen-coated 96-well flat-bottom plates (Falcon). Cells were then left under basal conditions or stimulated with cytokines for 24 hours (10 ng/ml recombinant tumor necrosis factor (TNF)-α, TNF-β, IL-1β, or oncostatin M all from PeproTech, London, UK) or lipopolysaccharide (Sigma). For all stimulations the cells were incubated in the presence or absence of IFN-γ (10 ng/ml, PeproTech). After stimulation supernatant from relevant wells was pooled and stored at −70°C until analyzed and the cell monolayers fixed in methanol. Nonspecific binding of mAb was inhibited by preincubation of cells for 1 hour at 37°C with 4% goat serum or rabbit serum as required (Sigma) before the addition of mouse anti-human mAb [ICAM-1: M7063, 1.85 μg/ml; VCAM-1: M7106, 1.75 μg/ml; E-selectin: M7105, 1.6 μg/ml; CD31: M0823, 2.25 μg/ml; cytokeratin 19: M0888, 0.5 μg/ml (all from DAKO) and ICAM-2: sc-1512, 1 μg/ml (Autogen Bioclear UK Ltd., Wiltshire, UK)] for 1 hour at 37°C. The cells were then washed thoroughly before incubation with peroxidase-conjugated goat anti-mouse or rabbit anti-goat secondary Ab as required (P0447 and P0449, respectively, 1/5000; DAKO). The enzyme-linked immunosorbent assay (ELISA) was developed using O-phenylenediamine substrate (OPD: S2045; DAKO) according to the manufacturer's instructions and the enzymatic reaction was stopped using 0.5 mol/L H2SO4 (Fisher Scientific, Lei-cestershire, UK). Colorimetric analysis was performed by measuring absorbance values at 490 nm using a Dynatech Laboratories MRX plate reader. All treatments were performed in triplicate for each experiment. The concentration of CXCL9, −10, and −11 in supernatant samples was determined by sandwich ELISA31Burdick MD Kunkel SL Lincoln PM Wilke CA Strieter RM Specific ELISAs for the detection of human macrophage inflammatory protein-1 alpha and beta.Immunol Invest. 1993; 22: 441-449Crossref PubMed Scopus (27) Google Scholar using commercially available kits (R&D Systems). Briefly, relevant capture antibodies were prepared in 0.1 mol/L carbonate/bicarbonate buffer, pH 9.6, added to 96-well immunosorp plates (Nunc) and allowed to coat overnight at 4°C. Protein standards of known concentration or test samples were then added to relevant wells and incubated at room temperature for 1 hour before washing and a further 1-hour incubation with relevant biotinylated detection antibodies. The plates were then washed thoroughly before incubation with peroxidase-conjugated streptavidin (1/5000; Zymed, Cambs, UK). The ELISA was developed using TMB substrate [100 μg/ml 3,5,3,5-tetramethylbenzidine (TMB; Sigma) in 0.11 mol/L sodium acetate, pH 5.5, and 0.0003% H2O2 (both BDH)] and the enzymatic reaction stopped using 2.5 mol/L H2SO4. Colorimetric analysis was performed by measuring absorbance values at 450 nm using a Dynatech Laboratories MRX plate reader. All measurements were performed in duplicate for each experiment. Lymphocytes isolated from chronically inflamed liver samples or matched peripheral blood samples were resuspended to a count of 8 × 104/ml. Eighteen-well Teflon-coated slides (Erie Scientific, Portsmouth, NH) were incubated with rhICAM-1 (5 μg/ml), rhVCAM-1 (5 μg/ml), or bovine serum albumin (1 μg/ml) and then incubated with CXCL9, −10, or −11 at 400 ng/ml for 90 minutes at 37°C. Some lymphocytes were preincubated with pertussis toxin (PTX), 100 ng/ml, to inhibit G-protein-coupled signaling. In control wells adhesion was triggered by the addition of the integrin-activator MnCl2 (100 nmol/L/ml). After nonadherent cells were removed slides were fixed and mounted before counting adherent lymphocytes in three representative high-power fields per well. To study the pattern of lymphocyte adhesion under the influence of physiological blood flow, HSECs were cultured to confluence in glass capillary tubes, stimulated for 24 hours with TNF-α and IFN-γ (both 10 ng/ml; R&D Systems) and connected to the flow-based system as described previously.29Lalor PF Clements JM Pigott R Humphries MJ Spragg JH Nash GB Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.Eur J Immunol. 1997; 27: 1422-1426Crossref PubMed Scopus (37) Google Scholar LILs were perfused through microslides at wall shear stresses relevant to those found in the liver sinusoids (0.05 Pa). Adherent cells were visualized microscopically under phase contrast and a video link was used to record assays for off-line analysis at a later date. The number of adherent cells was converted to adherent cells per millimeter squared and corrected for the number of cells perfused. The pattern of adhesion was analyzed to determine the number of cells rolling, statically adherent, or transmigrated. Cells appearing phase bright were above the endothelial monolayer whereas those appearing phase dark had migrated through the monolayer. For studies requiring the addition of exogenous chemokine the initial HSEC culture time was extended to allow development of cell surface glycosaminoglycans. HSECs were stimulated with TNF-α alone (10 ng/ml) 24 hours before assay and exogenous chemokine added for 15 minutes immediately before assay. For functional studies HSEC monolayers were incubated with blocking antibodies raised against ICAM-1 (11C81: 10 μg/ml) and VCAM-1 (4B2: 10 μg/ml) (both R&D Systems) or lymphocytes were incubated with a blocking antibody raised against CXCR3 (1C6: 10 μg/ml; kind gift of M. Briskin, Millennium Pharmaceuticals Inc., USA) or pertussis toxin (100 ng/ml; Sigma Aldrich Ltd., Aldrich). Adhesion of CXCR3-expressing lymphocytes to VCAM-1 in the presence of CXCL9, −10, and −11 was performed by first immobilizing recombinant human VCAM-1 (5 μg/ml; R&D Systems) in microslides followed by recombinant human chemokine at a variety of concentrations. Incubations were for 90 minutes at 37°C. Fresh tissue was homogenized in loading sample buffer, normalized for total protein using Coomassie blue, and loaded on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. After electrophoresis and transfer onto Hybond membranes (Amersham-Pharmacia) blocked with 10% skimmed milk, CXCR3 ligands were detected using mouse anti-human mAbs [CXCL9: 49106, 5 μg/ml; CXCL11: 87328, 5 μg/ml (both R & D Systems), CXCL10: 6D4. 5 μg/ml (Abcam, Cambs, UK)]. Rabbit anti-mouse horseradish peroxidase-conjugated antibody was used as a secondary step and demonstrated using the ECL detection system (Amersham Pharmacia). CXCL9, −10, and −11 chemokine gene expressions were analyzed by RT-PCR amplification of RNA from HSECs, comparing stimulated and unstimulated cells. Total RNA was extracted from cells using RNAzol extraction (Biogenesis, Bournemouth, UK), and single-stranded cDNA synthesis was conducted as previously described.32Chomsczynzki P Sacchi N Single-step method of RNA isolation by acid guadinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159PubMed Google Scholar The PCR reaction was conducted using 1 μl of cDNA per reaction. Reaction conditions were 1× (95°C, 5 minutes), 1× (55°C, 5 minutes), 35× (94°C, 30 seconds; 55°C, 30 seconds; 72°C, 2 minutes), and 1× (72°C, 5 minutes). Primers were designed from GenBank sequences for β-actin, CXCL9, CXCL10 and CXCL11: β-actin forward, 5′-CAT CAC CAT TGG CAA TGA GC-3′; β-actin reverse, 5′-CGA TCC ACA CGG AGT ACT TG-3′; CXCL9 forward, 5′-GGC AAC CAG ACC ATT GTC TC-3′; CXCL9 reverse, 5′-TTC TGG CCA CAG ACA ACC TC-3′; CXCL10 forward, 5′-CAG AAT CGA CCA TCA AG-3′; CXCL10 reverse, 5′-GGC AGT GGA AGT CCA TGA AG-3′; CXCL11 forward, 5′-AAC AGC ACC AGC AGC AAC AG-3′; CXCL11 reverse 5′-GTG CAC TGT TGC CAG TAT CC. Product sizes for each set of primers were: β-actin, 284 bp; CXCL9, 643 bp; CXCL10, 506 bp; CXCL11, 1041 bp. Positive and negative controls were included in each assay. Amplified products were analyzed on 2% agarose gel containing ethidium bromide. t-Tests (paired or independent) were used to assess data normally distributed whereas nonnormally distributed data were compared using Wilcoxon signed ranks test (for related samples) or Mann-Whitney U-tests (for unrelated samples). P values ≤0.1 were indicated by (*), ≤0.01; (**), and ≤0.001 (***). Statistics were performed using the software package SPSS version 11.0 (SPSS, Chicago, IL). Cultured HSECs and BECs displayed typical cellular morphology in culture and were positive for characteristic phenotypic markers. Thus BECs stained positively for the epithelial markers HEA-125 and cytokeratin 19, but were negative for CD31. HSECs stained positively for antibodies raise
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