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Protective Role of Interleukin-17 in Murine NKT Cell-Driven Acute Experimental Hepatitis

2010; Elsevier BV; Volume: 177; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2010.100028

1525-2191

Zenebech Wondimu, Tania Santodomingo-Garzón, Tai Le, Mark G. Swain,

T-cell and B-cell Immunology

NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1+ and CD4+ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis. NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1+ and CD4+ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis. The cytokine interleukin-17A (IL-17) has been increasingly identified as an important regulator of the inflammatory response.1Miossec P Korn T Kuchroo VK Interleukin-17 and type 17 helper T cells.N Engl J Med. 2009; 361: 888-898Crossref PubMed Scopus (1154) Google Scholar, 2Harrington LE Hatton RD Mangan PR Turner H Murphy TL Murphy KM Weaver CT Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages.Nat Immunol. 2005; 6: 1123-1132Crossref PubMed Scopus (3801) Google Scholar, 3Park H Li Z Yang XO Chang SH Nurieva R Wang YH Wang Y Hood L Zhu Z Tian Q Dong C A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol. 2005; 6: 1133-1141Crossref PubMed Scopus (3445) Google Scholar Initially, a new subset of CD4+ T cells were considered to be the source of IL-17 and were classified as Th17 cells.2Harrington LE Hatton RD Mangan PR Turner H Murphy TL Murphy KM Weaver CT Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages.Nat Immunol. 2005; 6: 1123-1132Crossref PubMed Scopus (3801) Google Scholar, 3Park H Li Z Yang XO Chang SH Nurieva R Wang YH Wang Y Hood L Zhu Z Tian Q Dong C A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol. 2005; 6: 1133-1141Crossref PubMed Scopus (3445) Google Scholar IL-17 secreted from Th17 cells was implicated as a proinflammatory mediator in a number of experimental models of inflammation, especially those associated with autoimmunity and an adaptive immune response.4Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3244) Google Scholar, 5Lubberts E Koenders MI Oppers-Walgreen B van den Bersselaar L Coenen-de Roo CJ Joosten LA van den Berg WB Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion.Arthritis Rheum. 2004; 50: 650-659Crossref PubMed Scopus (610) Google Scholar, 6Komiyama Y Nakae S Matsuki T Nambu A Ishigame H Kakuta S Sudo K Iwakura Y IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis.J Immunol. 2006; 177: 566-573PubMed Google Scholar However, more recently IL-17 has also been shown to be able to suppress inflammatory responses, mainly in experimental models which are characterized by a more pronounced innate immune response. Specifically, IL-17 has been shown to suppress inflammation in experimental murine models of asthma,7Schnyder-Candrian S Togbe D Couillin I Mercier I Brombacher F Quesniaux V Fossiez F Ryffel B Schnyder B Interleukin-17 is a negative regulator of established allergic asthma.J Exp Med. 2006; 203: 2715-2725Crossref PubMed Scopus (488) Google Scholar gastritis,8Otani K Watanabe T Tanigawa T Okazaki H Yamagami H Watanabe K Tominaga K Fujiwara Y Oshitani N Arakawa T Anti-inflammatory effects of IL-17A on Helicobacter pylori-induced gastritis.Biochem Biophys Res Commun. 2009; 382: 252-258Crossref PubMed Scopus (44) Google Scholar colitis,9Ogawa A Andoh A Araki Y Bamba T Fujiyama Y Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice.Clin Immunol. 2004; 110: 55-62Crossref PubMed Scopus (382) Google Scholar, 10O'Connor Jr, W Kamanaka M Booth CJ Town T Nakae S Iwakura Y Kolls JK Flavell RA A protective function for interleukin 17A in T cell-mediated intestinal inflammation.Nat Immunol. 2009; 10: 603-609Crossref PubMed Scopus (624) Google Scholar and atherosclerosis.11Taleb S Romain M Ramkhelawon B Uyttenhove C Pasterkamp G Herbin O Esposito B Perez N Yasukawa H Van Snick J Yoshimura A Tedgui A Mallat Z Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis.J Exp Med. 2009; 206: 2067-2077Crossref PubMed Scopus (324) Google Scholar However, the role of IL-17 in regulating hepatic inflammation remains unclear. In patients with viral hepatitis, alcoholic liver disease, and autoimmune liver diseases, numbers of IL-17-producing hepatic T cells are increased.12Lemmers A Moreno C Gustot T Marechal R Degre D Demetter P de Nadai P Geerts A Quertinmont E Vercruysse V Le Moine O Deviere J The interleukin-17 pathway is involved in human alcoholic liver disease.Hepatology. 2009; 49: 646-657Crossref PubMed Scopus (293) Google Scholar In murine models of liver inflammation the role of IL-17 in regulating the inflammatory response remains controversial. In murine T-cell-mediated hepatitis induced by concanavalin A administration, IL-17 has been shown to be both proinflammatory, as well as without a direct inflammation modulating role.13Zenewicz LA Yancopoulos GD Valenzuela DM Murphy AJ Karow M Flavell RA Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation.Immunity. 2007; 27: 647-659Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 14Nagata T McKinley L Peschon JJ Alcorn JF Aujla SJ Kolls JK Requirement of IL-17RA in Con A induced hepatitis and negative regulation of IL-17 production in mouse T cells.J Immunol. 2008; 181: 7473-7479PubMed Google Scholar NKT cells are an important component of the innate immune response and are highly enriched within the liver.15Swain MG Hepatic NKT cells: friend or foe?.Clin Sci (Lond). 2008; 114: 457-466Crossref PubMed Scopus (66) Google Scholar NKT cells are activated by glycolipid antigens presented in association with the major histocompatibility complex class I–like molecule CD1d expressed on the surface of antigen presenting cells.16Bendelac A Savage PB Teyton L The biology of NKT cells.Annu Rev Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar Activation of NKT cells in this fashion results in the rapid production and release of large amounts of both Th1; eg, interferon (IFN) γ, tumor necrosis factor (TNF) α, and Th2 (eg, IL-4) cytokines.16Bendelac A Savage PB Teyton L The biology of NKT cells.Annu Rev Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar NKT cells have been implicated in human liver disease and are of critical importance in the initiation and development of hepatitis in numerous murine models.15Swain MG Hepatic NKT cells: friend or foe?.Clin Sci (Lond). 2008; 114: 457-466Crossref PubMed Scopus (66) Google Scholar, 17Takeda K Hayakawa Y Van Kaer L Matsuda H Yagita H Okumura K Critical contribution of liver natural killer T cells to a murine model of hepatitis.Proc Natl Acad Sci USA. 2000; 97: 5498-5503Crossref PubMed Scopus (491) Google Scholar, 18Ajuebor MN Aspinall AI Zhou F Le T Yang Y Urbanski SJ Sidobre S Kronenberg M Hogaboam CM Swain MG Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells.J Immunol. 2005; 174: 8027-8037PubMed Google Scholar More recently, NKT cells have also been shown to be capable of rapidly producing IL-17 after activation.19Michel ML Keller AC Paget C Fujio M Trottein F Savage PB Wong CH Schneider E Dy M Leite-de-Moraes MC Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia.J Exp Med. 2007; 204: 995-1001Crossref PubMed Scopus (505) Google Scholar, 20Michel ML Mendes-da-Cruz D Keller AC Lochner M Schneider E Dy M Eberl G Leite-de-Moraes MC Critical role of ROR-gamma in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation.Proc Natl Acad Sci USA. 2008; 105: 19845-19850Crossref PubMed Scopus (202) Google Scholar, 21Yoshiga Y Goto D Segawa S Ohnishi Y Matsumoto I Ito S Tsutsumi A Taniguchi M Sumida T Invariant NKT cells produce IL-17 through IL-23-dependent and -independent pathways with potential modulation of Th17 response in collagen-induced arthritis.Int J Mol Med. 2008; 22: 369-374PubMed Google Scholar To date IL-17 has been reported to be produced mainly by type II (ie, non-invariant) and NK1.1 negative NKT cells19Michel ML Keller AC Paget C Fujio M Trottein F Savage PB Wong CH Schneider E Dy M Leite-de-Moraes MC Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia.J Exp Med. 2007; 204: 995-1001Crossref PubMed Scopus (505) Google Scholar, 22Lee KA Kang MH Lee YS Kim YJ Kim DH Ko HJ Kang CY A distinct subset of natural killer T cells produces IL-17, contributing to airway infiltration of neutrophils but not to airway hyperreactivity.Cell Immunol. 2008; 251: 50-55Crossref PubMed Scopus (51) Google Scholar, 23Coquet JM Chakravarti S Kyparissoudis K McNab FW Pitt LA McKenzie BS Berzins SP Smyth MJ Godfrey DI Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1.1- NKT cell population.Proc Natl Acad Sci USA. 2008; 105: 11287-11292Crossref PubMed Scopus (355) Google Scholar; however, within the murine liver most NKT cells express CD4 and NK1.1 and are classified as invariant (iNKT) or type I NKT cells.15Swain MG Hepatic NKT cells: friend or foe?.Clin Sci (Lond). 2008; 114: 457-466Crossref PubMed Scopus (66) Google Scholar, 16Bendelac A Savage PB Teyton L The biology of NKT cells.Annu Rev Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar α-Galactosylceramide (αGalCer) is a glycolipid, originally isolated from a marine sponge, which specifically activates iNKT cells in both humans and mice after being presented by antigen presenting cells in the context of CD1d.16Bendelac A Savage PB Teyton L The biology of NKT cells.Annu Rev Immunol. 2007; 25: 297-336Crossref PubMed Scopus (1784) Google Scholar iNKT cells activated in this fashion can in turn transactivate numerous other cell types within the liver, including other components of the innate immune response such as macrophages and NK cells.24Nakagawa R Nagafune I Tazunoki Y Ehara H Tomura H Iijima R Motoki K Kamishohara M Seki S Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584PubMed Google Scholar, 25Ajuebor MN Wondimu Z Hogaboam CM Le T Proudfoot AE Swain MG CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis.Am J Pathol. 2007; 170: 1975-1988Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar This property of αGalCer has generated interest in developing this compound as an immune stimulating agent for the treatment of human disease, including liver cancers.24Nakagawa R Nagafune I Tazunoki Y Ehara H Tomura H Iijima R Motoki K Kamishohara M Seki S Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice.J Immunol. 2001; 166: 6578-6584PubMed Google Scholar However, αGalCer treatment also induces hepatitis in mice and therefore has been used as an experimental model to study hepatic immune and inflammatory responses which result from the specific activation of iNKT cells and the subsequent downstream stimulation of the hepatic innate immune system.26Osman Y Kawamura T Naito T Takeda K Van Kaer L Okumura K Abo T Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide.Eur J Immunol. 2000; 30: 1919-1928Crossref PubMed Scopus (242) Google Scholar, 27Biburger M Tiegs G Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells.J Immunol. 2005; 175: 1540-1550PubMed Google Scholar Therefore, we undertook this series of experiments to determine first whether hepatic NK1.1 positive iNKT cells could also produce IL-17 after specific activation. In addition, given that the adaptive Th17 response develops more slowly, we wanted to determine the role of IL-17, released as part of the early iNKT cell–driven innate hepatic immune response, in the regulation of hepatitis induced by the administration of αGalCer. Male C57BL/6 mice were used (8–10 weeks old; The Jackson Laboratories, Bar Harbor, ME). All procedures were approved by the Animal Care Committee of the University of Calgary (protocol M07028) and were performed in accordance with the guidelines of the Canadian Council on Animal Care. The following reagents and antibodies were obtained from indicated sources: α-galactosylceramide (αGalCer, Alexis Biochemicals, San Diego, CA), collagenase type 2 (Cedarlane Laboratories, Burlington, ON, Canada), DNase I (Roche Diagnostics, Laval, Quebec, Canada), PBS57 (analog of α-GalCer) loaded CD1d tetramer conjugated to phycoerythrin (PE; kindly provided by National Institute of Allergy and Infectious Diseases, National Institutes of Health Tetramer Core Facility, Atlanta, GA). Purified or fluorescent conjugated monoclonal antibodies (mAbs) were purchased from the following suppliers: PerCp anti-CD4 (RM4-5), Percp-Cy5.5 anti-NK1.1 (PK136), fluorescein isothiocyanate (FITC) anti-TNFα (MP6-XT22), FITC or PE anti-CD11b (M1/70), PE anti-IL-12 (P40/P70, C15.6), PE anti IL-17A (TC11–18H10), anti-CD16/CD32 (2.4G2; BD Biosciences, Mississauga, ON, Canada); PE-Cy5 anti-F4/80 (BM8), FITC anti-Ly-6G (Gr1, RB6–8C5), FITC anti-IL-17 (eBio1757; eBioscience Inc, San Diego, CA); anti-Ly-6C (ER-MP20) and goat anti-rat IgG-FITC (Santa Cruz Biotechnology Inc., Santa Cruz, CA); PE-anti-IFNγ (XMG1.2) (BD Biosciences); PE-anti-IL-10 (JES5-16E3; eBioscience); PE anti-IL-4 (BVD4-1D11; BD Biosciences); primary anti-CCR2 (CKR-2B; Santa Cruz); and bovine anti-goat IgG PE secondary antibody (sc-3747; Santa Cruz); FITC annexin V (catalog no. 556419, BD Biosciences); anti-CCL2 (LS-C71953; LifeSpan Biosciences, Seattle, WA); and anti-CXCL5 (500-P146; PreproTech, Inc, Rocky Hill, NJ). To induce hepatitis, a single intravenous injection of αGalCer (2 μg in 100 μl vehicle; 2% DMSO and 0.04% Tween 20 in sterile PBS was administered per mouse.26Osman Y Kawamura T Naito T Takeda K Van Kaer L Okumura K Abo T Activation of hepatic NKT cells and subsequent liver injury following administration of alpha-galactosylceramide.Eur J Immunol. 2000; 30: 1919-1928Crossref PubMed Scopus (242) Google Scholar, 27Biburger M Tiegs G Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells.J Immunol. 2005; 175: 1540-1550PubMed Google Scholar Controls received 100 μl of vehicle. For IL-17 neutralization, a single dose of either 75 μg per mouse anti-mouse IL-17 mAb, or ratIgG2a isotype control, was injected intravenously 1 hour before αGalCer administration [anti-mouse IL-17 neutralizing mAb (MAB421) and rat IgG isotype control (MAB006); R&D Systems, Minneapolis, MN].10O'Connor Jr, W Kamanaka M Booth CJ Town T Nakae S Iwakura Y Kolls JK Flavell RA A protective function for interleukin 17A in T cell-mediated intestinal inflammation.Nat Immunol. 2009; 10: 603-609Crossref PubMed Scopus (624) Google Scholar, 28Hofstetter HH Ibrahim SM Koczan D Kruse N Weishaupt A Toyka KV Gold R Therapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis.Cell Immunol. 2005; 237: 123-130Crossref PubMed Scopus (360) Google Scholar To confirm important findings obtained with the R&D Systems neutralizing anti-IL-17 antibody (MAB421), we repeated some of the experiments with a second commercially available IL-17 neutralizing monoclonal antibody targeted to a different IL-17 epitope than the R&D Systems IL-17 neutralizing antibody (LEAF purified anti-mouse IL-17 neutralizing mAb (TC11-18H10.1) and LEAF purified rat IgG1 isotype control (RTK2071); BioLegend, San Diego, CA).29Zhang Z Clarke TB Weiser JN Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice.J Clin Invest. 2009; 119: 1899-1909PubMed Google Scholar In additional experiments the effects of exogenous recombinant murine (rm) IL-17 administration on αGalCer-induced hepatitis severity was determined [rmIL-17 (421-ML/CF); R&D Systems, Minneapolis, MN].11Taleb S Romain M Ramkhelawon B Uyttenhove C Pasterkamp G Herbin O Esposito B Perez N Yasukawa H Van Snick J Yoshimura A Tedgui A Mallat Z Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis.J Exp Med. 2009; 206: 2067-2077Crossref PubMed Scopus (324) Google Scholar For these experiments rmIL-17, 1 μg/mouse, or PBS vehicle was injected intraperitoneally 30 minutes before and 1 hour after αGalCer administration.8Otani K Watanabe T Tanigawa T Okazaki H Yamagami H Watanabe K Tominaga K Fujiwara Y Oshitani N Arakawa T Anti-inflammatory effects of IL-17A on Helicobacter pylori-induced gastritis.Biochem Biophys Res Commun. 2009; 382: 252-258Crossref PubMed Scopus (44) Google Scholar Initially, at 2, 8, 16, 24, and 48 hours after αGalCer or vehicle treatment mice were sacrificed and serum collected and livers perfused through the portal vein with sterile ice-cold PBS. To assess the degree of hepatic injury, blood was collected and alanine aminotransferase (ALT) levels measured (commercial kit; Biotron Diagnostics, Hemet, CA), and liver tissue samples collected in 10% buffered formalin for histological examination after H&E staining. αGalCer-induced hepatic injury is associated with a predominant innate immune response characterized by the early activation of iNKT cells and the subsequent accumulation of neutrophils and monocytes within the liver.30Delarbre C Gachelin G Injection of the immuno-modulatory drug alpha-galactosylceramide results in the recruitment of a large population of antigen-presenting cells into the liver of C57BL/6 mice.Microbes Infect. 2004; 6: 360-368Crossref PubMed Scopus (4) Google Scholar Therefore, we determined recruited neutrophil and monocyte positioning within the liver by immunohistochemistry at 16 hours after αGalCer treatment. Neutrophil recruitment to the liver was assessed by nonspecific esterase (Leder) staining of formalin fixed paraffin embedded liver tissue sections as previously described.31Bonder CS Ajuebor MN Zbytnuik LD Kubes P Swain MG Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis.J Immunol. 2004; 172: 45-53PubMed Google Scholar Monocyte recruitment to the liver was assessed by immunohistochemical staining of Ly-6C antigen (a cell surface marker highly expressed on infiltrating monocytes but not on resident tissue macrophages including Kupffer cells)32Geissmann F Jung S Littman DR Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar in formalin-fixed paraffin-embedded liver sections.33Whiteland JL Nicholls SM Shimeld C Easty DL Williams NA Hill TJ Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.J Histochem Cytochem. 1995; 43: 313-320Crossref PubMed Scopus (100) Google Scholar Endogenous peroxidase and endogenous biotin binding were blocked using 3% H2O2 and an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), respectively, and then stained with anti Ly-6C mAb. The bound antibody was detected by the peroxidase-labeled avidin-biotin complex method and diaminobenzidine (Fast DAB, Sigma) was used as a substrate for color development. Sections were then counterstained with Gills II hematoxylin. In addition, at specified time points after αGalCer or vehicle administration liver tissue was collected in Trizol reagent (Invitrogen, Burlington, ON, Canada) for RNA isolation or treated with digestion buffer (0.05% collagenase 2 and 0.02% DNase I in Hanks' balanced salt solution with Ca2+ and Mg2+ ions) for cell isolation. Hepatic mononuclear cells were isolated by discontinuous Percoll gradient (GE HealthCare Biosciences, Baue D'urfe, Quebec, Canada) as previously described.18Ajuebor MN Aspinall AI Zhou F Le T Yang Y Urbanski SJ Sidobre S Kronenberg M Hogaboam CM Swain MG Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells.J Immunol. 2005; 174: 8027-8037PubMed Google Scholar, 25Ajuebor MN Wondimu Z Hogaboam CM Le T Proudfoot AE Swain MG CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis.Am J Pathol. 2007; 170: 1975-1988Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Cell viability was assessed by Trypan Blue dye exclusion. Single cell suspensions (0.5–1.0 × 106 cells per sample) were prepared in binding buffer (1% fetal bovine serum in PBS) for flow cytometric staining. Plasma and livers were collected 2, 8, 16, and 24 hours after αGalCer or vehicle treatment and IL-17 levels were measured using a commercial enzyme-linked immunosorbent assay (BioLegend, San Diego, CA). For hepatic IL-17 measurements livers were perfused (as above) and whole livers were homogenized in 2 ml of buffer containing protease inhibitors, centrifuged, and filtered as described previously in detail.18Ajuebor MN Aspinall AI Zhou F Le T Yang Y Urbanski SJ Sidobre S Kronenberg M Hogaboam CM Swain MG Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells.J Immunol. 2005; 174: 8027-8037PubMed Google Scholar, 25Ajuebor MN Wondimu Z Hogaboam CM Le T Proudfoot AE Swain MG CCR5 deficiency drives enhanced natural killer cell trafficking to and activation within the liver in murine T cell-mediated hepatitis.Am J Pathol. 2007; 170: 1975-1988Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Immunophenotyping and intracellular cytokine detection were done by direct immunofluorescence using multicolor flow cytometry staining of isolated hepatic mononuclear cells, or total leukocytes, as previously described.18Ajuebor MN Aspinall AI Zhou F Le T Yang Y Urbanski SJ Sidobre S Kronenberg M Hogaboam CM Swain MG Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells.J Immunol. 2005; 174: 8027-8037PubMed Google Scholar FCγ III/II receptors were blocked by incubating isolated cells with anti-CD16/CD32. Hepatic NKT cells were identified by simultaneous staining with anti-CD3 or -CD4-PerCp or -NK1.1-FITC and CD1d-PBS57/PE; hepatic monocytes and neutrophils were identified by simultaneous staining with anti-F4/80-PE-Cy5 and -Ly-6G(Gr1)-FITC. In additional experiments monocytes were alternatively identified by staining with rat anti-mouse Ly-6C mAb and detected with a secondary goat anti-rat IgG-FITC. Tissue macrophages, including Kupffer cells, are Ly-6C negative.32Geissmann F Jung S Littman DR Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar For intracellular cytokine detection, cells were stained with mAbs to respective cell surface antigens and were then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) and stained with anti-IL-17-FITC, -IL-17-PE, or -TNFα-FITC, or -IL-12-PE, or -IFNγ-PE, or -IL-4-PE, or -IL-10-PE, or -annexin V-FITC. Cell surface CCR2 expression was determined using an unconjugated anti-CCR2 and PE-labeled secondary antibodies. Stained cells were acquired and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest Alias software. iNKT cells producing IL-17 were identified by gating on NK1.1 or CD4 and CD1d-PBS57 double positive cells that were positive for IL-17. Similarly, monocytes producing IL-12, TNFα, or IL-10 were identified by gating on cells that were double positive for F4/80 and IL-12, IL-10, or TNFα. Infiltration of inflammatory cells into the tissues is regulated by chemokines.34Charo IF Ransohoff RM The many roles of chemokines and chemokine receptors in inflammation.N Engl J Med. 2006; 354: 610-621Crossref PubMed Scopus (2011) Google Scholar The chemokines CCL2/MCP-1 or CXCL1/KC, CXCL2/MIP-2 and CXCL5/LIX have been directly implicated in the recruitment of monocytes and neutrophils, respectively, into tissues during an inflammatory response.35Jaeschke H Chemokines and liver inflammation: the battle between pro- and anti-inflammatory mediators.Hepatology. 1997; 25: 252-253Crossref PubMed Scopus (10) Google Scholar, 36Shields 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 Therefore, we determined the hepatic expression of these four chemokines using real-time PCR in mice pretreated with IgG or anti-IL-17 neutralizing antibody and sacrificed 2 hours after αGalCer treatment. Total RNA was extracted from 100 mg of liver tissue frozen in Trizol reagent (Invitrogen Canada Inc., Burlington, ON, Canada) from IgG control and anti-IL-17-treated mice according to standard protocols.37Overbergh L Giulietti A Valckx D Decallonne R Bouillon R Mathieu C The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression.J Biomol Tech. 2003; 14: 33-43PubMed Google Scholar The hepatic expressions of CCL2/MCP-1, CXCL1/KC, CXCL2/MIP-2 and CXCL5/LIX mRNAs (primers from SABiosciences, Frederick, MD) were analyzed by real-time quantitative PCR using RT2 SYBR Green/ROX qPCR Master Mix (SABiosciences). Data were analyzed with ABI Prism 7000 SDS software (Applied Biosystems) using a relative quantification method according to the manufacturer's protocol. Expression levels of the target genes were normalized to endogenous GAPDH expression. Data are presented as fold-increase in hepatic chemokine mRNA expression relative to levels determined for vehicle-treated mice. To complement our mRNA findings, hepatic CXCL5 and CCL2 protein expression was determined by immunohistochemistry, using previously described methods,38Van Lint P Wielockx B Puimege L Noel A Lopez-Otin C Libert C Resistance of collagenase-2 (matrix metalloproteinase-8)-deficient mice to TNF-induced lethal hepatitis.J Immunol. 2005; 175: 7642-7649PubMed Google Scholar, 39Hammerschmidt E Loeffler I Wolf G Morg1 heterozygous mice are protected from acute renal ischemia-reperfusion injury.Am J Physiol Renal Physiol. 2009; 297: F1273-F1287Crossref PubMed Scopus (23) Google Scholar in formalin fixed paraffin-embedded liver sections obtained from anti-IL-17-pretreated mice